Evidence for new nuclear and mitochondrial genome organizations among high-frequency somatic embryogenesis-derived plants of allotetraploid Coffea arabica L. (Rubiaceae)

 The most important commercial species of coffee, Coffea arabica, which produces 73% of the world's coffee crop and almost all of the coffee in Latin America, is the only tetraploid (allotetraploid, 2n=4x=44) species known in the genus. High-frequency somatic embryogenesis, plant regeneration and plant recovery were achieved from leaf explants of a mature, elite plant of C. arabica cv. Cauvery (S-4347) using a two-step culture method. To assess the genetic integrity of the nuclear, mitochondrial and chloroplast genomes among the hardened regenerants, we employed multiple DNA markers (RFLP, RAPD, ISSR) for sampling various regions of the genome. Although the nuclear and mitochondrial genomes of the mother plant and five ramets derived from the mother ortet were similar in organization, this was not so in the somatic embryo-derived plants where both nuclear and mitochondrial genomes changed in different, characteristic ways and produced novel genome organizations. A total of 480 genetic loci, based on the data obtained from a total of 16 nuclear, mitochondrial and chloroplast gene probes, in combination with nine restriction enzyme digests, 38 RAPD and 17 SSR primers, were scored in 27 somatic embryo-derived plants and the single control. Among these, 44 loci were observed to be polymorphic. A relatively low level of polymorphism (4.36%) was found in the nuclear genome, while polymorphism in the mitochondrial genome (41%) was much higher. No polymorphism was detected in the chloroplast genome. The polymorphism in the mitochondrial genome was found in only 4 plants. Such selective polymorphism was not true for the nuclear genome. Thus, this in-depth and comprehensive study demonstrates, for the first time, the presence of subtle genetic variability and novel genome organizations in the commercially well-established somatic embryogenesis-derived plants of this important coffee species. Received: 2 July 1999 / Revision received: 1 February 2000 / Accepted: 17 February 2000     

Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination

Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.     

Updating Phylogeny of Mitochondrial DNA Macrohaplogroup M in India: Dispersal of Modern Human in South Asian Corridor

To construct maternal phylogeny and prehistoric dispersals of modern human being in the Indian sub continent, a diverse subset of 641 complete mitochondrial DNA (mtDNA) genomes belonging to macrohaplogroup M was chosen from a total collection of 2,783 control-region sequences, sampled from 26 selected tribal populations of India. On the basis of complete mtDNA sequencing, we identified 12 new haplogroups - M53 to M64; redefined/ascertained and characterized haplogroups M2, M3, M4, M5, M6, M8′C′Z, M9, M10, M11, M12-G, D, M18, M30, M33, M35, M37, M38, M39, M40, M41, M43, M45 and M49, which were previously described by control and/or coding-region polymorphisms. Our results indicate that the mtDNA lineages reported in the present study (except East Asian lineages M8′C′Z, M9, M10, M11, M12-G, D ) are restricted to Indian region.The deep rooted lineages of macrohaplogroup ‘M’ suggest in-situ origin of these haplogroups in India. Most of these deep rooting lineages are represented by multiple ethnic/linguist groups of India. Hierarchical analysis of molecular variation (AMOVA) shows substantial subdivisions among the tribes of India (Fst = 0.16164). The current Indian mtDNA gene pool was shaped by the initial settlers and was galvanized by minor events of gene flow from the east and west to the restricted zones. Northeast Indian mtDNA pool harbors region specific lineages, other Indian lineages and East Asian lineages. We also suggest the establishment of an East Asian gene in North East India through admixture rather than replacement.     

‘Bite-and-Switch’ approach using computationally designed molecularly imprinted polymers for sensing of creatinine

A method for the selective detection of creatinine is reported, which is based on the reaction between polymerised hemithioacetal, formed by allyl mercaptan, o-phthalic aldehyde, and primary amine leading to the formation of fluorescent isoindole complex. This method has been demonstrated previously for the detection of creatine using creatine-imprinted molecularly imprinted polymers (MIPs) Since MIPs created using traditional methods were unable to differentiate between creatine and creatinine, a new approach to the rational design of a molecularly imprinted polymer (MIP) selective for creatinine was developed using computer simulation. A virtual library of functional monomers was assigned and screened against the target molecule, creatinine, using molecular modelling software. The monomers giving the highest binding score were further tested using simulated annealing in order to mimic the complexation of the functional monomers with template in the monomer mixture. The result of this simulation gave an optimised MIP composition. The computationally designed polymer demonstrated superior selectivity in comparison to the polymer prepared using traditional approach, a detection limit of 25 μM and good stability. The ‘Bite-and-Switch’ approach combined with molecular imprinting can be used for the design of assays and sensors, selective for amino containing substances.     

The role of dynamic stimulation pattern in the analysis of bistable intracellular networks

Bistable systems play an important role in the functioning of living cells. Depending on the strength of the necessary positive feedback one can distinguish between (irreversible) “one-way switch” or (reversible) “toggle-switch” type behavior. Besides the well- established steady-state properties, some important characteristics of bistable systems arise from an analysis of their dynamics. We demonstrate that a supercritical stimulus amplitude is not sufficient to move the system from the lower (off-state) to the higher branch (on-state) for either a step or a pulse input. A switching surface is identified for the system as a function of the initial condition, input pulse amplitude and duration (a supercritical signal). We introduce the concept of bounded autonomy for single level systems with a pulse input. Towards this end, we investigate and characterize the role of the duration of the stimulus. Furthermore we show, that a minimal signal power is also necessary to change the steady state of the bistable system. This limiting signal power is independent of the applied stimulus and is determined only by systems parameters. These results are relevant for the design of experiments, where it is often difficult to create a defined pattern for the stimulus. Furthermore, intracellular processes, like receptor internalization, do manipulate the level of stimulus such that level and duration of the stimulus is conducive to characteristic behavior.     

Amelogenin-deficient mice display an amelogenesis imperfecta phenotype.

Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human X-chromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.     

Complex systems biology approach to understanding coordination of JAK-STAT signaling

In this work, we search for coordination as an organizing principle in a complex signaling system using a multilevel hierarchical paradigm. The objective is to explain the underlying mechanism of Interferon (IFN_γ) induced JAK-STAT (specifically JAK1/JAK2-STAT1) pathway behavior. Starting with a mathematical model of the pathway from the literature, we modularize the system using biological knowledge via principles of biochemical cohesion, biological significance, and functionality. The modularized system is then used as a basis for in silico inhibition, knockdown/deletion and perturbation experiments to discover a coordination mechanism. Our analysis shows that a module representing the SOCS1 complex can be identified as the coordinator. Analysis of the coordinator can then be used for the selection of biological experiments for the discovery of ‘soft’ molecular drug targets, that could lead to the development of improved therapeutics. The coordinator identified is also being investigated to determine its relationship to pathological conditions.     

Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N

The use of 2-L polyethylene terephthalate beverage bottles as a bacterial culture vessel has been recently introduced as an enabling technology for high-throughput structural biology [Sanville Millard, C. et al., 2003. Protein Express. Purif. 29, 311-320]. In the article following this one [Stols et al., this issue, pp. 95-102], this approach was elaborated for selenomethionine labeling used for multiwavelength anomalous dispersion phasing in the X-ray crystallographic determinations of protein structure. Herein, we report an effective and reproducible schedule for uniform 15N- and 13C-labeling of recombinant proteins in 2-L beverage bottles for structural determination by NMR spectroscopy. As an example, three target proteins selected from Arabidopsis thaliana were expressed in Escherichia coli Rosetta (DE3)/pLysS from a T7-based expression vector, purified, and characterized by electrospray ionization mass spectrometry and NMR analysis by 1H-15N heteronuclear single quantum correlation spectroscopy. The results show that expressions in the unlabeled medium provide a suitable control for estimation of the level of production of the labeled protein. Mass spectral characterizations show that the purified proteins contained a level of isotopic incorporation equivalent to the isotopically labeled materials initially present in the growth medium, while NMR analysis of the [U-15N]-labeled proteins provided a convenient method to assess the solution state properties of the target protein prior to production of a more costly double-labeled sample.