Extension and use of a physical map of the Thinopyrum-derived Lr19 translocation

Twenty-nine deletion mutant lines were used to extend a physical map of the Lr19 translocated chromosome segment. One hundred and forty-four Sse8387I/MseI and 32 EcoRI/MseI primer combinations were used to obtain 95 Thinopyrum-specific AFLP markers. The physical map confirmed that terminal deletions had mostly occurred, however, it appears that intercalary deletions and primer or restriction site mutations were also induced. The markers allowed for grouping of the deletion mutant lines into 19 clusters, with 7 AFLP markers mapping in the same marker bin as Lr19. Primary and secondary Lr19 allosyndetic recombinants were subsequently physically mapped employing AFLP, RFLP, SCAR and microsatellite markers and the data integrated with the deletion map. A further shortened, tertiary Lr19 recombinant was derived following homologous recombination between the proximally shortest secondary recombinant, Lr19-149-299, and distally shortest recombinant, Lr19-149-478. The tertiary recombinant could be confirmed employing the mapped markers and it was possible to identify new markers on this recombinant that can be used to reduce the translocation still further.     

Ca2+-dependent lipid binding and membrane integration of PopA, a harpin-like elicitor of the hypersensitive response in tobacco

PopA is released by type III secretion from the bacterial plant pathogen Ralstonia solanacearum and triggers the hypersensitive response (HR) in tobacco. The function of PopA remains obscure, mainly because mutants lacking this protein are not altered in their ability to interact with plants. In an attempt to identify the site of PopA activity in plant cells, we generated transgenic tobacco plants expressing the popA gene under the control of an inducible promoter. Immunocytologic analysis revealed that the HR phenotype of these plants correlated with the presence of PopA at the plant plasma membrane. Membrane localization was observed irrespective of whether the protein was designed to accumulate in the cytoplasm or to be secreted by the plant cell, suggesting a general lipid-binding ability. We found that the protein had a high affinity for sterols and sphingolipids in vitro and that it required Ca2+ for both lipid binding and oligomerization. In addition, the protein was integrated into liposomes and membranes from Xenopus laevis oocytes where it formed ion-conducting pores. These characteristics suggest that PopA is part of a system that aims to attach the host cell plasma membrane and to allow molecules cross this barrier.     

Gas exchange characteristics, metabolic rate and water loss of the Heelwalker, Karoophasma biedouwensis (Mantophasmatodea: Austrophasmatidae)

This study presents the first physiological information for a member of the wingless Mantophasmatodea, or Heelwalkers. This species shows cyclic gas exchange with no evidence of a Flutter period (more typical of discontinuous gas exchange in insects) and no indication that the spiracles are fully occluded during quiescent metabolism. Standard metabolic rate at 20 degrees C was 21.32+/-2.73 microl CO(2)h(-1) (mean+/-S.E.), with a Q(10) (10-25 degrees C) of 1.7. Increases in V()CO(2) associated with variation in mass and with trial temperature were modulated by an increase in burst period volume and a decline in cycle frequency. Total water loss rate, determined by infrared gas analysis, was 0.876+/-0.08 mg H(2)Oh(-1) (range 0.602-1.577, n=11) whilst cuticular water loss rate, estimated by linear regression of total water loss rate and metabolic rate, was 0.618+/-0.09 mg H(2)Oh(-1) (range 0.341-1.363, n=11). Respiratory water loss rate was therefore no more than 29% of the total rate of water loss. Both total water loss rate and estimated cuticular water loss rate were significantly repeatable, with intraclass correlation coefficients of 0.745 and 0.553, respectively.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Structural,Functional, and Evolutionary Analysis of the Unusually Large Stilbene Synthase Gene Family in Grapevine

Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.Plants produce a vast array of secondary metabolites, many of them being restricted to specific groups of plant species. This extraordinary chemical diversity is believed to have evolved from a limited number of ubiquitous biosynthetic pathways through gene duplication followed by functional divergence (Pichersky and Gang, 2000). The phenylpropanoid pathway, derived from Phe, illustrates perfectly this phenomenon, as it gives rise to a large diversity of phenolic compounds playing key roles in plants, including participation in structural polymers, defense against herbivores and pathogens, protection from abiotic stress, and important functions in plant-pollinator interactions. Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including dicotyledon angiosperms such as grapevine (Vitis vinifera), peanut (Arachis hypogaea), and Japanese knotweed (Fallopia japonica, formerly Polygonum cuspidatum), monocotyledons like sorghum (Sorghum bicolor), and gymnosperms such as several Pinus and Picea species. In addition to their participation in both constitutive and inducible defense mechanisms in plants, several stilbenes display important pharmacological properties. Since resveratrol (3,5,4′-trihydroxy-trans-stilbene) was postulated to be involved in the health benefits associated with a moderate consumption of red wine (Renaud and de Lorgeril, 1992), plant stilbenes have received considerable interest. Nowadays, resveratrol ranks among the most extensively studied natural products, and hundreds of studies have shown that it can slow the progression of a wide variety of illnesses, including cancer and cardiovascular disease, as well as extend the life spans of various organisms (Baur and Sinclair, 2006). Stilbene synthases (STSs) are characteristic of stilbene-producing plants and catalyze the biosynthesis of the stilbene backbone from three malonyl-CoA and one CoA-ester of a cinnamic acid derivative. STSs are members of the type III polyketide synthases family, chalcone synthases (CHSs), which catalyze the first step of flavonoid biosynthesis, being the most ubiquitous polyketide synthase in plants. Both CHS and STS use p-coumaroyl-CoA and malonyl-CoA as substrates and synthesize the same linear tetraketide intermediate. However, STS uses a specific cyclization mechanism involving a decarboxylation to form the stilbene backbone. STS proteins share extensive amino acid sequence identity with CHS, and phylogenetic analysis of the STS and CHS gene families has shown that STS genes may have evolved from CHS genes several times independently (Tropf et al., 1994). In most stilbene-producing plants, STS genes form small families of closely related paralogs. For example, two STS cDNAs have been cloned from peanut (Schröder et al., 1988), the genome of Scots pine (Pinus sylvestris) has been shown to contain a small family of four STS genes (Preisig-Müller et al., 1999), and three STS genes have been characterized in Japanese red pine (Pinus densiflora; Kodan et al., 2002). Only one STS gene has been isolated from Japanese knotweed to date (Liu et al., 2011), and the sequencing of sorghum genome has shown that SbSTS1 was the only STS gene in this plant species (Yu et al., 2005; Paterson et al., 2009). Grapevine is a noteworthy exception among stilbene-producing plants, as its genome has been shown to contain a large family of putative STS genes. Early Southern-blot experiments suggested that the grapevine genome contained more than 20 STS genes (Sparvoli et al., 1994). Analyses of the first drafts of the grapevine genome sequence confirmed the large size of this multigene family, with an estimated number of STS genes ranging from 21 to 43 (Jaillon et al., 2007; Velasco et al., 2007). However, these relatively low-coverage sequence drafts did not allow a precise analysis of large families of highly similar genes. The more recently released 12× genome sequence of grapevine inbred Pinot Noir cultivar PN40024 offered an improved sequence quality, allowing an accurate analysis of the STS gene family. In this work, we take advantage of the improved 12× sequence of the grapevine ‘PN40024’ genome to analyze the grapevine STS gene family. Furthermore, we combine molecular evolution to structural and functional analyses to gain more insight into the significance of the remarkable amplification of the STS family in grapevine.     

Rapid de novo evolution of X chromosome dosage compensation in Silene latifolia, a plant with young sex chromosomes

Silene latifolia is a dioecious plant with heteromorphic sex chromosomes that have originated only ～10 million years ago and is a promising model organism to study sex chromosome evolution in plants. Previous work suggests that S. latifolia XY chromosomes have gradually stopped recombining and the Y chromosome is undergoing degeneration as in animal sex chromosomes. However, this work has been limited by the paucity of sex-linked genes available. Here, we used 35 Gb of RNA-seq data from multiple males (XY) and females (XX) of an S. latifolia inbred line to detect sex-linked SNPs and identified more than 1,700 sex-linked contigs (with X-linked and Y-linked alleles). Analyses using known sex-linked and autosomal genes, together with simulations indicate that these newly identified sex-linked contigs are reliable. Using read numbers, we then estimated expression levels of X-linked and Y-linked alleles in males and found an overall trend of reduced expression of Y-linked alleles, consistent with a widespread ongoing degeneration of the S. latifolia Y chromosome. By comparing expression intensities of X-linked alleles in males and females, we found that X-linked allele expression increases as Y-linked allele expression decreases in males, which makes expression of sex-linked contigs similar in both sexes. This phenomenon is known as dosage compensation and has so far only been observed in evolutionary old animal sex chromosome systems. Our results suggest that dosage compensation has evolved in plants and that it can quickly evolve de novo after the origin of sex chromosomes.     

A new brace treatment similar for adolescent scoliosis and kyphosis based on restoration of thoracolumbar lordosis. Radiological and subjective clinical results after at least one year of treatment

Study design

A prospective treatment study with a new brace was conducted Objective. To evaluate radiological and subjective clinical results after one year conservative brace treatment with pressure onto lordosis at the thoracolumbar joint in children with scoliosis and kyphosis.

Summary of background data

Conservative brace treatment of adolescent scoliosis is not proven to be effective in terms of lasting correction. Conservative treatment in kyphotic deformities may lead to satisfactory correction. None of the brace or casting techniques is based on sagittal forces only applied at the thoracolumbar spine (TLI= thoracolumbar lordotic intervention). Previously we showed in patients with scoliosis after forced lordosis at the thoracolumbar spine a radiological instantaneous reduction in both coronal curves of double major scoliosis.

Methods

A consecutive series of 91 children with adolescent scoliosis and kyphosis were treated with a modified symmetric 30 degrees Boston brace to ensure only forced lordosis at the thoracolumbar spine. Scoliosis was defined with a Cobb angle of at least one of the curves [greater than or equal to] 25 degrees and kyphosis with or without a curve <25 degrees in the coronal plane. Standing radiographs were made i) at start, ii) in brace at beginning and iii) after one year treatment without brace.

Results

Before treatment start ??in brace?? radiographs showed a strong reduction of the Cobb angles in different curves in kyphosis and scoliosis groups (sagittal n = 5 all p < 0.001, pelvic obliquity p < 0.001). After one year of brace treatment in scoliosis and kyphosis group the measurements on radiographs made without brace revealed an improvement in 3 Cobb angles each.

Conclusion

Conservative treatment using thoracolumbar lordotic intervention in scoliotic and kyphotic deformities in adolescence demonstrates a marked improvement after one year also in clinical and postural criteria. An effect not obtained with current brace techniques.     

Mutation rate and genome reduction in endosymbiotic and free-living bacteria

Genome reduction has been considered the hallmark of endosymbiotic bacteria, such as endocellular mutualists or obligatory pathogens until it was found exactly the same in several free-living bacteria. In endosymbiotic bacteria genome reduction is mainly attributed to degenerative processes due to small population size. These cannot affect the free-living bacteria with reduced genomes because they are known to have very large population sizes. It has been proposed that selection for simplification drove genome reduction in these free-living bacteria. For at least one of them (Prochlorococcus), genome reduction is associated with accelerated evolution and we suggest an alternative hypothesis based on increase in mutation rate as the primary cause of genome reduction in free-living bacteria.