Native and artificial forisomes: functions and applications

Forisomes are remarkable protein bodies found exclusively in the phloem of the Fabaceae. When the phloem is wounded, forisomes are converted from a condensed to a dispersed state in an ATP-independent reaction triggered by Ca²⁺, thereby plugging the sieve tubes and preventing the loss of photoassimilates. Potentially, forisomes are ideal biomaterials for technical devices because the conformational changes can be replicated in vitro and are fully reversible over a large number of cycles. However, the development of technical devices based on forisomes has been hampered by the laborious and time-consuming process of purifying native forisomes from plants. More recently, the problem has been overcome by the production of recombinant artificial forisomes. This is a milestone in the development of forisome-based devices, not only because large quantities of homogeneous forisomes can be produced on demand, but also because their properties can be tailored for particular applications. In this review, we discuss the physical and molecular properties of native and artificial forisomes, focusing on their current applications in technical devices and potential developments in the future.     

Micromechanical measurements on P-protein aggregates (forisomes) from Vicia faba plants

Forisomes are chemomechanically active P-protein aggregates found in the phloem of legumes. They can convert chemical energy into mechanical work when induced by divalent metal ions or changes in pH, which control the folding state of individual forisome proteins. We investigated the changing geometric parameters of individual forisomes and the strength and dynamics of the forces generated during this process. Three different divalent ions were tested (Ca²⁺, Sr²⁺ and Ba²⁺) and were shown to induce similar changes to the normalized length and diameter. In the concentration range from 0.1 to 4 M, K⁺ and Cl^? ions had no influence on the contraction behaviour of the forisomes induced by 10 mM Ca²⁺. In the absence of dissolved oxygen, these changes were independent of the radius of the metal ion, water uptake and the strength of binding between the selected metal ions and those protein molecules responsible for forisome conformational transformation. In the absence of any load, bound Ca²⁺, Sr²⁺ and Ba²⁺ ions showed apparent and averaged dissociation constants of 14, 62 and 1070 µM, respectively. Various forisomes generated bending on a quartz glass fibre with a diameter of 9 µm. The fibre bending was measured microscopically also by correlation between the digital patterns of a predefined window of observation before and after bending. Similar bending forces of approximately 90 nN were measured for a single forisome sequentially exposed to 10 mM Ca²⁺, Sr²⁺ and Ba²⁺. In the absence of dissolved oxygen, the same conditions resulted in averaged bending forces of (93 ± 40) nN, (58 ± 20) nN, and (91 ± 20) nN after contacting different forisomes with 10 mM Ca²⁺, 10 mM Sr²⁺, and 10 mM Ba²⁺ respectively, demonstrating that the force generated was independent on ion concentrations above a certain threshold value.     

Transfer of yeast artificial chromosomes from yeast to mammalian cells.

Human DNA can be cloned as yeast artificial chromosomes (YACs), each of which contains several hundred kilobases of human DNA. This DNA can be manipulated in the yeast host using homologous recombination and yeast selectable markers. In relatively few steps it is possible to make virtually any change in the cloned human DNA from single base pair changes to deletions and insertions. In order to study the function of the cloned DNA and the effects of the changes made in the yeast, the human DNA must be transferred back into mammalian cells. Recent experiments indicate that large genes can be transferred from the yeast host to mammalian cells in tissue culture and that the genes are transferred intact and are expressed. Using the same methods it may soon be possible to transfer YAC DNA into the mouse germ line so that the expression and function of genes cloned in YACs can be studied in developing and adult mammalian animals.     

Recombinant artificial forisomes provide ample quantities of smart biomaterials for use in technical devices

Forisomes are mechanoproteins that undergo ATP-independent contraction–expansion cycles triggered by divalent cations, pH changes, and electrical stimuli. Although native forisomes from Medicago truncatula comprise a number of subunits encoded by separate genes, here we show that at least two of those subunits (MtSEO1 and MtSEO4) can assemble into homomeric forisome bodies that are functionally similar to their native, multimeric counterparts. We expressed these subunits in plants and yeast, resulting in the purification of large quantities of artificial forisomes with unique characteristics depending on the expression platform. These artificial forisomes were able to contract and expand in vitro like native forisomes and could respond to electrical stimulation when immobilized between interdigital transducer electrodes. These results indicate that recombinant artificial forisomes with specific characteristics can be prepared in large amounts and used as components of microscale and nanoscale devices.     

Linnaeus's natural and artificial arrangements of plants

Linnaeus's artificial and natural arrangements of plants are examined using a Spearman rank coefficient (which is explained) on his presentations of his own and others' arrangements in the Classes plantarum and elsewhere. There is little alteration in his successive artificial arrangements. In contrast, between 1751 and 1764 his natural arrangements changed considerably, partly in the sequences of genera within orders but mostly by rearrangement of the orders. Comparison with Cesalpino's and Ray's natural arrangements, using the longest-recognized natural groups as signposts, suggests that Linnaeus in his latest natural arrangement (1764) approximated more closely to Ray's. Examination of Linnaeus's successive treatments of certain groups (palms, Zingiberaceae, Hydrocharis-Stratiotes-Vallisneria) and of Giseke's exposition of Linnaeus's lectures on natural groups (1792) shows that Linnaeus was much influenced by habitus and vegetative characters as well as those of the fructification. He recognized orders consisting of a chain of genera linked successively by overall affinity and without any single diagnostic character. Where possible, he preferred characters of the fructification and his ‘secret’ consulting of the habitus is explained as secondary to such characters. It is suggested that in his latest arrangement he approximated more to a scala naturae, as he probably did in zoology about the same time. Within his artificial arrangements Linnaeus kept to sequences of genera as natural as possible. He realized that some groups in his natural arrangements were still artificial, and his aphorism that all genera and species are natural, classes and orders part natural and part artificial, refers to his and others' practice until the natural system could be completed. It is not a statement of the essential natures of these ranks. Linnaeus's distinction in practice between natural and artificial arrangements was less clear-cut than Sachs believed. Linnaeus's rejection of the ancient tree/herb division was empirical, not a reasoned repudiation of an a priori grouping. The tree/herb division could be upheld in his day as obviously natural, not merely accepted on authority.     

Expression of an artificial yeast TRP-gene cluster in yeast and Escherichia coli

Summary All five tryptophan biosynthetic genes of Saccharomyces cerevisiae were unified on plasmid pME554, which is based on 2 m DNA and pBR322 sequences allowing for autonomous replication in yeast and E. coli. Homologous and heterologous expression of this artificial yeast TRP-gene cluster was studied. Plasmid pME554 allowed for nearly normal growth of a yeast strain bearing auxotrophic mutations in all five TRP-genes. The plasmid-borne genes TRP2 to TRP5 were expressed and regulated normally in the frame of the general control. Gene TRP1, carried on an EcoRI/BglII fragment lacking the ARS1 function, was expressed poorly and did not respond to the general control like the chromosomally-borne TRP1 gene.Plasmid pME554 allowed for poor growth of E. coli strain W3110 tna ^– trpEA2 on minimal medium. Marked stimulation was observed, however, when anthranilic acid or indole were added. Accordingly, poor expression of the first Trp-enzyme anthranilate synthase and the last enzyme tryptophan synthase was found, whereas the other three genes were moderately well expressed in E. coli.Abbreviations bp basepair - kb kilobase     

PCR amplification and analysis of yeast artificial chromosomes

A strategy for the analysis of yeast artificial chromosome (YAC) clones that relies on polymerase chain reaction (PCR) amplification of small restriction fragments from isolated YACs following adapter ligation was developed. Using this method, termed YACadapt, we have amplified several YACs from a human Xq24-qter library and have used the PCR products for physical mapping by somatic cell hybrid deletion analysis and fluorescent in situ hybridization. One YAC, RS46, was mapped to band Xq27.3, near the fragile X mutation. The PCR product is an excellent renewable source of YAC DNA for analyses involving hybridization of YAC inserts to a variety of DNA/RNA sources.     

Making artificial honey using yeast cells from salivary glands of honey bees

The salivary glands of a honey bee, Apis cerana and the yeast cells isolated from these glands were studied for their effects on sucrose solution. This solution exhibited lowered pH and increased levels of fructose and total amino acids as the time of incubation proceeded. The solution thus made was similar to the natural honey.     

High-efficiency yeast artificial chromosome fragmentation vectors

Chromosome fragmentation vectors (CFVs) are used to create deletion derivatives of large fragments of human DNA cloned as yeast artificial chromosomes (YACs). CFVs target insertion of a telomere sequence into the YAC via homologous recombination with Alu repetitive elements. This event results in the loss of all YAC sequences distal to the site of integration. A new series of CFVs has been developed. These vectors target fragmentation to both Alu and LINE human repetitive DNA elements. Recovery of deletion derivatives is ten- to 20-fold more efficient with the new vectors than with those described previously.     

Transgenic mice carrying yeast artificial chromosomes

The generation of transgenic mice with yeast artificial chromosomes (YACs) has proven to be a valuable system to: (1) study gene structure-function relationships; (2) produce mouse models of human disease; (3) complement mouse mutants; (4) generate mice bioreactors; and (5) screen YAC libraries in vivo. Continued refinement of current techniques and development of new protocols should encourage widespread adaptation of this strategy for these and other applications. Use of whole loci as transgenes is an important improvement in murine transgenesis because it results in a more realistic pattern and level of gene expression during ontogeny. Application of this technology to develop human artificial chromosomes (HACs) might provide the next generation of gene therapy vectors that will overcome most of the problems and barriers associated with current vector systems.     

Characteristics of immobilized yeast reactors producing ethanol from glucose

The evolutionary performance of up flow reactors are affected by the cell immobilization matrix and the matrix particle size distribution. Higher productivities are obtained using a low-density brick with a particle size of ca. 400-1400 mum. A medium condition favoring growth quickly leads to large biomass gradients within the reactors and, eventually, reductions in average productivities due to bed plugging. These systems can be accurately modelled using Monod kinetics when dispersion and the biomass gradient are considered. The productivity was apparently not controlled by substrate diffusion in these cases.     

Mitotic recombination of yeast artificial chromosomes.

Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments in vivo by meiotic recombination. This is of importance for reconstruction of a long gene or a gene complex. In this work we have taken advantage of yeast protoplast fusion to generate isosexual diploids followed by mitotic crossing-over, and show that it can be an alternative simple strategy for recombining YACs. Integrative transformation of one of the parent strains with the construct pRAN4 (containing the ADE2 gene) is used to disrupt the URA3 gene contained within the pYAC4 vector arm, providing the markers required for forcing fusion and detecting recombination. All steps can be carried out within the commonly used AB1380 host strain without the requirement for micromanipulation. The method was applied to YAC clones from the human MHC and resulted in the reconstruction of a 650 kb long single clone containing 18 known genes from the MHC class II region.     

Vesicle fusion and fission in plants and yeast

Measurements of the membrane capacitance on animal cells has provided an excellent technique for monitoring of exo- and endocytotic activity in intact living cells. Here we review recent data in which the same technique was applied to plant cells and cells of the budding yeast Saccharomyces cerevisiae. The data show that unitary exo- and endocytotic events can also be measured with the same technique after removing the cell wall from these cells. The resulting protoplasts execute the same type of transient and permanent fusion/fission that is known from animal cells. Also the size of the vesicles, which are fusing or budding, are of the same order of magnitude as those recorded in animal cells. Together these data support the view of an evolutionary conserved mechanism for unitary exo- and endocytosis events in eukaryotes. The successful recordings of exo- and endocytotic activity in Saccharomyces cerevisiae by capacitance measurements now pave the way for correlating the abundant information on the molecular machinery of exo- and endocytosis in this model organism with distinct functional properties.     

Construction and characterization of a partial library of yeast artificial chromosomes from human chromosome 21

We report a protocol for cloning large DNA fragments in yeast artificial chromosomes (YAC). A partial library has been constructed from a somatic hybrid containing chromosome 21 as the single source of human DNA. About 4.0 Mb of human DNA was recovered in 17 YAC clones. Three clones were analyzed by in situ hybridization and mapped on chromosome 21. One clone hybridized with the chromosome 21 centromeric region and may provide new insight both on the molecular structure of centromere and on the localization of Alzheimer disease gene.     

Effect of yeast extracts on higher plants

Summary Effect of yeast autolyzate on the growth of vineless pea plant was investigated.The yield was increased remarkably when yeast autolyzate was added, as compared with the control area in which the plants received only mineral fertilizer solution.The yield stimulation was the highest where 0.1 ppm of the autolyzate was added, the yield increasing by more than 80%. The reason why the yields were lower in the areas where the amount of yeast autolyzate added was incrased to 1 ppm and to 10 ppm, was presumed to be due to the fact that the substances may have been absorbed, decomposed, and utilized by soil microorganisms.