Cell genetic engineering: Transmission genetics of plants

Achievements of cell and genetic engineering that led to formation of a new genetics chapter— transmission genetics—have been described. Results have been analyzed and new opportunities in the field of transgenomic somatic hybrids and cybrid obtaining, production of transgenic plants with agronomic pharmaceutical application, development of transplastomic plants, and accumulation of recombinant proteins by using the transient expression of foreign genes in plants have been shown.     

Structure and Protein-Protein Interaction Studies on Chlamydia trachomatis Protein CT670 (YscO Homolog)

Comparative genomic studies have identified many proteins that are found only in various Chlamydiae species and exhibit no significant sequence similarity to any protein in organisms that do not belong to this group. The CT670 protein of Chlamydia trachomatis is one of the proteins whose genes are in one of the type III secretion gene clusters but whose cellular functions are not known. CT670 shares several characteristics with the YscO protein of Yersinia pestis, including the neighboring genes, size, charge, and secondary structure, but the structures and/or functions of these proteins remain to be determined. Although a BLAST search with CT670 did not identify YscO as a related protein, our analysis indicated that these two proteins exhibit significant sequence similarity. In this paper, we report that the CT670 crystal, solved at a resolution of 2 Å, consists of a single coiled coil containing just two long helices. Gel filtration and analytical ultracentrifugation studies showed that in solution CT670 exists in both monomeric and dimeric forms and that the monomer predominates at lower protein concentrations. We examined the interaction of CT670 with many type III secretion system-related proteins (viz., CT091, CT665, CT666, CT667, CT668, CT669, CT671, CT672, and CT673) by performing bacterial two-hybrid assays. In these experiments, CT670 was found to interact only with the CT671 protein (YscP homolog), whose gene is immediately downstream of ct670. A specific interaction between CT670 and CT671 was also observed when affinity chromatography pull-down experiments were performed. These results suggest that CT670 and CT671 are putative homologs of the YcoO and YscP proteins, respectively, and that they likely form a chaperone-effector pair.Chlamydiae are obligate intracellular bacteria that infect a variety of eukaryotes, including humans, animals, insects, and free-living amoebae (8, 31, 51). They are highly pathogenic and cause genital tract, ocular, and respiratory infections in humans (9, 55). A key characteristic of Chlamydiae is their biphasic developmental cycle, in which the bacteria alternate between two morphologies: the elementary body and the reticulate body (1, 31). The Chlamydiae species, like many other Gram-negative pathogenic bacteria, contain a type III secretion (T3S) system, which plays a major role in their pathogenicity (4, 7, 44). This key system aids pathogenicity by exporting bacterial proteins, termed effectors, into the host cell via a syringe-like nanomachine called an injectisome (4, 7). Once secreted into the host cell, these effectors may manipulate host cell functions to the advantage of the pathogen (44, 54).Because of the unique chlamydial developmental cycle currently there are no tractable methods for genetic manipulation of these organisms (25, 49). Detailed bioinformatic investigations have identified approximately 200 proteins unique to various taxonomic levels of the Chlamydiae phylum (19, 21). Included in this pool of proteins are proteins whose genes occur in chlamydial T3S system loci (23, 44). Most of the Chlamydiae-specific genes in these loci encode proteins whose functions are unknown or putative and are referred to as Chlamydiae-specific putative T3S-related proteins. These genes include the ct670 and ct671 genes, which are downstream of the gene for the ATPase of the T3S system (ct669) and upstream of yscQ (ct672). CT670 is a Chlamydiales-specific protein with no known homologs (based on BLAST searches) in other species outside this group (21). However, based on similarities in size, charge distribution, and predicted secondary structure, CT670 is thought to be the Chlamydia trachomatis equivalent of YscO, a mobile core component of the T3S system in Yersinia (40). Previous studies of CPn0706, the Chlamydophila pneumoniae homolog of CT670, indicated that this protein was localized in the inclusion in infected cells, but it was not detected in the inclusion membrane or host cytosol, suggesting that it is not a secreted protein (24). Moreover, CT670 is not expected to be a type III secreted effector on the basis of the results obtained by a computational approach that was used to predict type III secreted effectors by comparison of sequences to sequences of known effectors (47). CT671 is a Chlamydiaceae-specific protein with no known homologs in any other species outside this family (21), but it is predicted to be a homolog of YscP, which is the molecular ruler and substrate specificity switch in Yersinia species (2). This protein is secreted by a heterologous T3S system and was predicted to be a T3S effector using the computational approach mentioned above (47, 53). Furthermore, the CCA00037 protein, the CT671 homolog in Chlamydophila caviae, has been visualized in the host cytosol of C. caviae-infected cells using specific antibodies, which provided evidence that it is secreted (53). Although CT670 and CT671 do not appear to be related to YscO and YscP, respectively, on the basis of the results of BLAST searches, it is possible that they have similar roles in the chlamydial T3S system based on their genetic neighborhood and other characteristics noted above. Further, because of their Chlamydiae specificity, they may provide a unique characteristic of the chlamydial T3S system. To examine this possibility, detailed investigations of the three-dimensional (3D) structure of CT670, its self-association properties, and its binding partners were carried out in the present study. Here we report elucidation of the CT670 crystal structure at a resolution of 2.0 Å. CT670 crystallized as a monomer with an elongated two-helix coiled coil. Using analytical ultracentrifugation, CT670 was found to be mostly monomeric in solution, but a dimeric form was also detected. Furthermore, by performing protein-protein interaction studies involving bacterial two-hybrid assays, as well as biochemical experiments, we obtained evidence showing that CT670 interacts specifically with CT671, which would be expected if these proteins have functions similar to those of YscO and YscP, respectively.     

Lithium desensitizes brain mitochondria to calcium, antagonizes permeability transition, and diminishes cytochrome C release

Among the numerous effects of lithium on intracellular targets, its possible action on mitochondria remains poorly explored. In the experiments with suspension of isolated brain mitochondria, replacement of KCl by LiCl suppressed mitochondrial swelling, depolarization, and a release of cytochrome c induced by a single Ca2+ bolus. Li+ robustly protected individual brain mitochondria loaded with rhodamine 123 against Ca2+-induced depolarization. In the experiments with slow calcium infusion, replacement of KCl by LiCl in the incubation medium increased resilience of synaptic and nonsynaptic brain mitochondria as well as resilience of liver and heart mitochondria to the deleterious effect of Ca2+. In LiCl medium, mitochondria accumulated larger amounts of Ca2+ before they lost the ability to sequester Ca2+. However, lithium appeared to be ineffective if mitochondria were challenged by Sr2+ instead of Ca2+. Cyclosporin A, sanglifehrin A, and Mg2+, inhibitors of the mitochondrial permeability transition (mPT), increased mitochondrial Ca2+ capacity in KCl medium but failed to do so in LiCl medium. This suggests that the mPT might be a common target for Li+ and mPT inhibitors. In addition, lithium protected mitochondria against high Ca2+ in the presence of ATP, where cyclosporin A was reported to be ineffective. SB216763 and SB415286, inhibitors of glycogen synthase kinase-3beta, which is implicated in regulating reactive oxygen species-induced mPT in cardiac mitochondria, did not increase Ca2+ capacity of brain mitochondria. Altogether, these findings suggest that Li+ desensitizes mitochondria to elevated Ca2+ and diminishes cytochrome c release from brain mitochondria by antagonizing the Ca2+-induced mPT.     

Production of bakuchiol by in vitro systems of <Emphasis Type=Italic>Psoralea drupacea</Emphasis> Bge

In vitro production of the meroterpene bakuchiol by Psoralea drupacea Bge (Fabaceae) has been studied using aseptically-grown plants, callus cultures of different origin, cell suspensions and transgenic hairy root cultures. The effect of phytohormones and methyl jasmonate on bakuchiol production was also investigated. Bakuchiol was not detected in cell suspensions or hairy root preparations of P. drupacea. In contrast, aerial parts of P. drupacea grown in vitro were found to accumulate up to 11% dry weight of bakuchiol and can therefore be regarded as a potentially useful source of this antimicrobial compound.     

Stable transformation of Solanum rickii chloroplast DNA

The biolistic method was used for genetic transformation of Solanum rickii chloroplasts with aadA gene encoding resistance to streptomycine and spectinomycine. Selective pressure was applied immediately after microbombardment to avoid appearance of mutant lines. The transplastomic Solanum rickii plants remained green during two years cultivation on the media supplemented with two antibiotics. There were no morphological differences between the transformed and the wild type plants.     

Obtaining of hairy-root,callus and suspenison cell cultures of carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) able to accumulate human interferon alpha-2b

Here we report the obtaining of suspension, callus and hairy root culture initiated from carrot plants of Nantskaya and Perfektzya variety with the highest level of recombinant human interferon-2b accumulation exhibiting the highest level of plant protein extract antiviral activity (up to 12.8 × 10³ IU/mg TSP). The antiviral activity of callus extracts was significantly lower comparing to the activity of plant extracts from the parent organisms. However, the antiviral activity level of suspension culture extracts (up to 4.42 × 10³ IU/mg TSP) and Ri-root ones (up to 4.42 × 10³ IU/mg TSP) appeared to be comparable to analogical data of antiviral activity of transgenic carrot leaf extracts, this way the described cultures could be possibly used for comparatively speedy obtaining of recombinant therapeutic protein for curing and preventing of virus diseases.     

Thermodynamic invariants of gel to the liquid-crystal 1,2-diacylphosphatidylcholines transition

The bilayer phase transitions from the ripple gel phase (P'(β)) to the liquid-crystal phase (L(α)) of a series of 1,2-diacylphosphatidylcholines containing a linear saturated acyl chain (C=14-19) have been studied by high-pressure scanning microcalorimetry. It has been shown that at ambient pressure, the transition temperature increases non-linearly depending on the acyl chain length. Pressure stabilizes the gel phase of lipids in a similar way; the pressure derivatives of the logarithm transition temperature as function of pressure are identical for all lipids. Based on the results obtained it has been concluded that the ratio γ of volume to enthalpy increments upon transitions in 1,2-diacylphosphatidylcholines is not dependent on the acyl chain length. When pressure grows, this ratio decreases drastically remaining identical for all lipids studied. Besides it has been demonstrated that increments of coefficients of thermal expansibility and isothermal compressibility are also rigidly bound to each other. Semi-empirical equations permitting to estimate volume parameters of the gel-to-liquid transition for 1,2-diacylphosphatidylcholines are given. The reasons for invariance of γ are discussed.     

POSH acts as a scaffold for a multiprotein complex that mediates JNK activation in apoptosis

We report that the multidomain protein POSH (plenty of SH3s) acts as a scaffold for the JNK pathway of neuronal death. This pathway consists of a sequential cascade involving activated Rac1/Cdc42, mixed-lineage kinases (MLKs), MAP kinase kinases (MKKs) 4 and 7, c-Jun N-terminal kinases (JNKs) and c-Jun, and is required for neuronal death induced by various means including nerve growth factor (NGF) deprivation. In addition to binding GTP-Rac1 as described previously, we find that POSH binds MLKs both in vivo and in vitro, and complexes with MKKs 4 and 7 and with JNKs. POSH overexpression promotes apoptotic neuronal death and this is suppressed by dominant-negative forms of MLKs, MKK4/7 and c-Jun, and by an MLK inhibitor. Moreover, a POSH antisense oligonucleotide and a POSH small interfering RNA (siRNA) suppress c-Jun phosphorylation and neuronal apoptosis induced by NGF withdrawal. Thus, POSH appears to function as a scaffold in a multiprotein complex that links activated Rac1 and downstream elements of the JNK apoptotic cascade.