Free Molecule Studies by Perturbed γ-γ Angular Correlation: A New Path to Accurate Nuclear Quadrupole Moments.

Accurate nuclear quadrupole moment values are essential as benchmarks for nuclear structure models and for the interpretation of experimentally determined nuclear quadrupole interactions in terms of electronic and molecular structure. Here, we present a novel route to such data by combining perturbed γ-γ angular correlation measurements on free small linear molecules, realized for the first time within this work, with state-of-the-art ab initio electronic structure calculations of the electric field gradient at the probe site. This approach, also feasible for a series of other cases, is applied to Hg and Cd halides, resulting in Q(^{199}Hg,5/2^{-})=+0.674(17) b and Q(^{111}Cd,5/2^{+})=+0.664(7) b.

Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development.

Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single-cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra-embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two-cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single-cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre-implantation development, embryonic stem cells and the establishment of the germ line.

Annealing studies combined with low temperature emission Mössbauer spectroscopy of short-lived parent isotopes: Determination of local Debye-Waller factors.

An extension of the online implantation chamber used for emission Mössbauer Spectroscopy (eMS) at ISOLDE/CERN that allows for quick removal of samples for offline low temperature studies is briefly described. We demonstrate how online eMS data obtained during implantation at temperatures between 300 K and 650 K of short-lived parent isotopes combined with rapid cooling and offline eMS measurements during the decay of the parent isotope can give detailed information on the binding properties of the Mössbauer probe in the lattice. This approach has been applied to study the properties of Sn impurities in ZnO following implantation of 119In (T½ = 2.4 min). Sn in the 4+ and 2+ charge states is observed. Above T > 600 K, Sn2+ is observed and is ascribed to Sn on regular Zn sites, while Sn2+ detected at T < 600 K is due to Sn in local amorphous regions. A new annealing stage is reported at T ≈ 550 K, characterized by changes in the Sn4+ emission profile, and is attributed to the annihilation of close Frenkel pairs.

Local increase of the Curie temperature in Mn/Fe implanted Y3Fe5O12 (YIG).

The change in the Curie temperature of single crystalline garnet Y3Fe5O12 (YIG) sample due to lattice damage induced by ion implantation has been investigated in 57Fe emission Mössbauer Spectroscopy (eMS) following implantation of 57Mn (T½ = 1.5 min). The Mössbauer spectra analysis reveal high spin Fe3+ ions substituted on both the octahedral and the tetrahedral sites. Measurements in the temperature range 298 K-798 K show that average values of the magnetic hyperfine field are decreased by the implantation-induced damage on the local lattice structure of the YIG. The Curie temperature, however, is determined to be 651 ± 5 K, considerably higher than the value of bulk YIG (559 K). This is most likely due to lattice damage-induced changes on the spin configurations of YIG through a FeA-O-FeD distortion scheme.

Transgenic plants as tools to study the molecular organization of plant genes.

Transgenic plants are generated in nature by Agrobacterium tumefaciens, a pathogen that produces disease through the transfer of some of its own DNA into susceptible plants. The genes are carried on a plasmid. Much has been learned about how the plasmid is transferred, how the plasmid-borne genes are organized, regulated, and expressed, and how the bacteria's pathogenic effects are produced. The A. tumefaciens plasmid has been manipulated for use as a general vector for the transfer of specific segments of foreign DNA of interest (from plants and other sources) into plants; the activities of various genes and their regulation by enhancer and silencer sequences have been assessed. Future uses of the vector (or others like it that have different host ranges) by the agriculture industry are expected to aid in moving into vulnerable plants specific genes that will protect them from such killers as nonselective herbicides, insects, and viruses.

Modification of plant lipid synthesis.

Genetic engineering of new storage oils and fats has produced oil crop plants with fatty acid compositions unattainable by plant breeding alone. The combination of classical breeding methods with molecular techniques provides new ways for designing oils for food and nonfood uses. Alterations in the position and number of double bonds, variation in fatty acid chain length, and the introduction of desired functional groups have already been achieved in model systems. Short-term prospects include crops such as rapeseed or soybean engineered to have greater than 70 to 80 percent medium-chain fatty acids by content, greater than 90 percent oleic acid, and high erucic acid content, and engineered to form ricinoleic acid in seed storage tissues.

Activation of a plant gene by T-DNA tagging: auxin-independent growth in vitro.

A transferred DNA (T-DNA) tagging vector with the potential to produce dominant mutations was used with cocultured Agrobacterium tumefaciens and protoplasts to tag genes involved in the action of the plant growth substance auxin. Transgenic calli were selected for their ability to grow in the absence of auxin in the culture media. From one experiment, 12 calli that displayed this phenotype were recovered, of which 11 were able to regenerate into plants. In one plant studied in detail, protoplast division in the absence of auxin genetically cosegregated with a single T-DNA insert. A messenger RNA encoded by a 6.4-kilobase sequence of plant genomic DNA rescued from the mutant is overexpressed relative to untransformed plants. The genomic DNA, as well as a cognate complementary DNA, once transfected into protoplasts promote growth and cell division in vitro in the absence of exogenously added auxin.

Splicing of the rolA transcript of Agrobacterium rhizogenes in Arabidopsis.

The rolA gene encoded on the Ri plasmid A4 of Agrobacterium rhizogenes is one of the transferred (TL-DNA) genes involved in the pathogenesis of hairy-root disease in plants. The function of the 100-amino acid protein product of rolA is unknown, although its expression causes physiological and developmental alterations in transgenic plants. The rolA gene of A. rhizogenes contains an intron in its untranslated leader region that has features typical of plant pre-messenger RNA introns. Transcription and splicing of the rolA pre-messenger RNA occur in the plant cell.

Release of active cytokinin by a beta-glucosidase localized to the maize root meristem.

A beta-glucoside encoded by a cloned Zea mays complementary DNA (Zm-p60.1) cleaved the biologically inactive hormone conjugates cytokinin-O-glucosides and kinetin-N3-glucoside, releasing active cytokinin. Tobacco protoplasts that transiently expressed Zm-p60.1 could use the inactive cytokinin glucosides to initiate cell division. The ability of protoplasts to sustain growth in response to cytokinin glucosides persisted indefinitely after the likely disappearance of the expression vector. In the roots of maize seedlings, Zm-p60.1 was localized to the meristematic cells and may function in vivo to supply the developing maize embryo with active cytokinin.

Viviparous leaves produced by somatic activation of an inactive cytokinin-synthesizing gene.

Tobacco plants that are somatic mosaics for expression of a cytokinin-synthesizing gene have viviparous leaves. Such a formation of shoots in an abnormal position represents a significant deviation from the usual organization of the plant body where a central axis produces shoots only in the axils of lateral leaf appendages and according to a precise phyllotactic pattern. This report links vivipary to the expression of a gene whose product is involved in the synthesis of the phytohormone cytokinin.

Growth of tobacco protoplasts stimulated by synthetic lipo-chitooligosaccharides.

fat Nodulation (Nod) factors are lipo-chitooligosaccharides (LCOs) secreted by rhizobia to trigger the early steps of nodule organogenesis in leguminous plants. A method to synthesize LCOs in vitro was developed. Synthetic LCOs alleviated the requirement for auxin and cytokinin to sustain growth of cultured tobacco protoplasts. LCOs containing C(18:1) trans-fatty acyl substituents were more effective than those containing cis-fatty acids in promoting cell division as well as in activating an auxin-responsive promoter and the expression of a gene implicated in auxin action. These data indicate that LCOs redirect plant growth also in nonlegumes by activating developmental pathways also targeted by phytohormones.

Introduction of genetic material into plant cells.

The tumor-inducing (Ti) plasmid of the soil microorganism Agrobacterium tumefaciens is the agent of crown gall disease in dicotyledonous plants. The Ti plasmid contains two regions that are essential for the production of transformed cells. One of these regions, termed transfer DNA, induces tumor formation and is found in all established plant tumor lines; the other, termed the virulence region, is essential for the formation but not the maintenance of tumors. Transfer DNA, which transfers to the plant genomes in a somewhat predictable manner, can be increased in size by the insertion of foreign DNA without its transferring ability being affected. The tumor-causing genes can be removed so that they no longer interfere with normal plant growth and differentiation. This modified Ti plasmid can thus be used as a vector for the transfer of foreign genes into plants.

Tumor DNA structure in plant cells transformed by A. tumefaciens.

Crown gall tumors are induced in plants by infection with the soil bacterium Agrobacterium tumefaciens. Because the tumor induction involves transfer of a portion of the tumor-inducing (Ti) plasmid DNA from the bacterium to the plant cells, this system is of interest for the study of genetic exchange as well as tumor induction. The boundaries of the transferred DNA (T-DNA) have been cloned from transformed plant cells of tobacco. Detailed mapping with restriction enzymes and nucleotide sequence analysis of two independent clones were used to study the molecular structure of the ends of the T-DNA. One clone contains the two ends of the T-DNA joined together; the other contains one end of the T-DNA joined to repetitive plant DNA sequences. These studies provide direct evidence that the T-DNA can be integrated into the plant genome. In addition, the data suggest that in the plant, T-DNA can be tandemly repeated. Sequence analysis of the junction of crown gall clone 1 reveals several direct repeats as well as an inverted repeat; these structures may be involved in the transfer of the DNA from Agrobacterium to plant cells.

Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a nonlegume.

The gene ENOD40 is expressed during early stages of legume nodule development. A homolog was isolated from tobacco, which, as does ENOD40 from legumes, encodes an oligopeptide of about 10 amino acids. In tobacco protoplasts, these peptides change the response to auxin at concentrations as low as 10(-12) to 10(-16)M. The peptides encoded by ENOD40 appear to act as plant growth regulators.

Localization of Elements Important for the Wound-Inducible Expression of a Chimeric Potato Proteinase Inhibitor II-CAT Gene in Transgenic Tobacco Plants.

The effect of progressive 5[prime] deletions within a potato proteinase inhibitor II promoter on wound-inducible expression of the chloramphenicol acetyltransferase (CAT) gene in leaves of transgenic tobacco plants was analyzed. After deletion of a region ranging from position -1300 to -700 with respect to the transcription start site, promoter activity was markedly reduced but still wound-inducible. Further deletion of approximately 200 base pairs resulted in a promoter activity that was below the detection limit, proving that the activity of the proteinase inhibitor II promoter is controlled by sequences upstream of position -514. Addition of the enhancer of the 35S promoter of the cauliflower mosaic virus (CaMV) either 5[prime] upstream or 3[prime] downstream of chimeric genes consisting of different proteinase inhibitor II promoter deletions (-700, -514, -210) fused to the CAT gene led to wound-inducible CAT gene expression in a fraction of transgenic plants containing either the "-700" or "-514" promoters, indicating the presence of wound-responsive elements in the promoter-proximal region. A fragment of the proteinase inhibitor II promoter comprising sequences between positions -1300 and -195 is able to confer wound-inducible expression to an inactive CaMV 35S promoter truncated at position -90 in either orientation, proving that this fragment displays wound-specific, enhancer-like properties. In addition, data are presented excluding that the proteinase inhibitor II 3[prime] end is of importance for wound-inducible gene expression.

Developmental and UV Light Regulation of the Snapdragon Chalcone Synthase Promoter.

Expression directed by the 1.1-kb snapdragon chalcone synthase (CHS) promoter linked to the [beta]-glucuronidase reporter gene has been studied in transgenic tobacco. The pattern of expression of the chimeric gene was compared with the expression of the endogenous CHS genes in tobacco and snapdragon. We demonstrate that expression of the CHS promoter is controlled in both an organ-specific and tissue-specific manner. The highest level of expression was observed in immature seeds. Deletions were used to define regions of the promoter required for expression in roots, stems, leaves, seeds, and flower petals of transgenic plants. We have defined the minimal sequences required for expression in different organs and mapped regions of the promoter that influence expression in either a positive or negative manner. A promoter fragment truncated to -39 activates transcription in roots of 4-week-old seedlings, whereas a fragment extending to -197 bp directs expression in petals and seeds. A positive regulatory element located between -661 and -566 and comprising a 47-bp direct repeat is active in all tissues investigated except petals. UV light-regulated expression in leaves of transgenic tobacco seedlings is dependent on the presence of sequences also required for leaf-specific expression. Within the intact promoter, sequences that individually confer different patterns of expression interact to produce the highly regulated expression pattern of CHS.

Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells.

Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.

The impact of Ti-plasmid-derived gene vectors on the study of the mechanism of action of phytohormones.

The molecular basis of tumor formation on dicotyledonous plants by Agrobacterium relies on the transfer to the plant cell of a unique segment of bacterial DNA, the T-DNA. The T-DNA contains genes that are active in the plant cell and encode hormone biosynthetic enzymes, or proteins that deregulate the cell's response to phytohormones. Study of this process has yielded not only knowledge of how alterations in phytohormone homeostasis can affect plant cell growth, but also has provided the essential tools to study phytohormone signaling in transgenic plants. Furthermore, T-DNA insertion into the plant genome forms the basis of gene tagging, a versatile method for isolating genes involved in phytohormone signal transduction and action.

Transformed cell clones as a tool to study T-DNA integration mediated by Agrobacterium tumefaciens.

A large number of tobacco SR1 cell clones transformed by the wild-type Agrobacterium C58 have been analysed for the presence of screenable markers such as tumour morphology, opine synthesis and hormone dependence. Distinct phenotypic classes were observed depending upon whether the cell clones were isolated from primary tumours or were obtained via cocultivation of protoplasts. These classes of tobacco SR1-C58 transformants appear to arise from errors in the Ti plasmid (T-DNA) transfer and integration mechanism itself rather than from subsequent T-DNA rearrangements, since 900 subclones, obtained by recloning a wild-type SR1-C58-transformed cell clone, yielded no variation in the phenotypes. A detailed genomic T-DNA analysis showed the presence of characteristic, abnormally short T-DNAs in the teratoma-forming, Acs- class and also in the Nos- class. The abnormal right border in two Nos- clones ends close to a sequence that resembles the normal T-DNA terminus and lies adjacent to the nos promoter, suggesting that this sequence could have functioned as a recognition site directing these particular T-DNA transfers. On the basis of the phenotypic and genomic blotting data it is clear that the short T-DNAs are characteristic of the cocultivation method. Other phenomena causing phenotypic variation, such as the loss of the T-DNA, and the gradual repression of T-DNA gene expression by methylation, are the main causes of aberrations in primary tumours. Moreover, the physical data suggest that early in the transformation cycle of Agrobacterium a replication step of a preselected T-DNA occurs before integration into the plant genome.

Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium meliloti.

In Rhizobium meliloti, expression of the nodulation genes (nod and nol genes) is under both positive and negative controls. These genes are activated by the products of the three related nodD genes, in conjunction with signal molecules from the host plants. We showed that negative regulation is mediated by a repressor protein, binding to the overlapping nodD1 and nodA as well as to the nodD2 promoters. The encoding gene, termed nolR, was identified and cloned from strain 41. By subcloning, deletion and Tn5 mutagenesis, a region of 594 base-pairs was found to be necessary and sufficient for repressor production in strains of R. meliloti lacking the repressor or in Escherichia coli. Sequence analysis revealed that nolR encodes a 13,349 Da protein, which is in agreement with the molecular weight of the NolR protein, determined after purification by affinity chromatography, utilizing long synthetic DNA multimers of the 21 base-pair conserved repressor-binding sequence. Our data suggest that the native NolR binds to the operator site in dimeric form. The NolR contains a helix-turn-helix motif, which shows homology to the DNA-binding sequences of numerous prokaryotic regulatory proteins such as the repressor XylR or the activator NodD and other members of the LysR family. Comparison of the putative DNA-binding helix-turn-helix motifs of a large number of regulatory proteins pointed to a number of novel regularities in this sequence. Hybridizations with an internal nolR fragment showed that sequences homologous to the nolR gene are present in all R. meliloti isolates tested, even in those that do not produce the repressor. In another species, such as Rhizobium leguminosarum, where NodD is autoregulated, however, such sequences were not detected.