The N-terminal region of Arabidopsis cystathionine gamma-synthase plays an important regulatory role in methionine metabolism

Cystathionine gamma-synthase (CGS) is a key enzyme of Met biosynthesis in bacteria and plants. Aligning the amino acid sequences revealed that the plant enzyme has an extended N-terminal region that is not found in the bacterial enzyme. However, this region is not essential for the catalytic activity of this enzyme, as deduced from the complementation test of an Escherichia coli CGS mutant. To determine the function of this N-terminal region, we overexpressed full-length Arabidopsis CGS and its truncated version that lacks the N-terminal region in transgenic tobacco (Nicotiana tabacum) plants. Transgenic plants expressing both types of CGS had a significant higher level of Met, S-methyl-Met, and Met content in their proteins. However, although plants expressing full-length CGS showed the same phenotype and developmental pattern as wild-type plants, those expressing the truncated CGS showed a severely abnormal phenotype. These abnormal plants also emitted high levels of Met catabolic products, dimethyl sulfide and carbon disulfide. The level of ethylene, the Met-derived hormone, was 40 times higher than in wild-type plants. Since the alien CGS was expressed at comparable levels in both types of transgenic plants, we further suggest that post-translational modification(s) occurs in this N-terminal region, which regulate CGS and/or Met metabolism. More specifically, since the absence of the N-terminal region leads to an impaired Met metabolism, the results further suggest that this region plays a role in protecting plants from a high level of Met catabolic products such as ethylene.     

Site-directed rotational resonance solid-state NMR distance measurements probe structure and mechanism in the transmembrane domain of the serine bacterial chemoreceptor

The serine receptor of bacterial chemotaxis is an ideal system in which to investigate the molecular mechanism of transmembrane signaling. Solid-state nuclear magnetic resonance (NMR) techniques such as rotational resonance provide a means for measuring local structure and ligand-induced structural changes in intact membrane proteins bound to native membrane vesicles. A general site-directed biosynthetic (13)C labeling strategy is used to direct the distance measurements to a specific site; the distance is measured between a unique Cys residue and a non-unique, low-abundance residue (Tyr or Phe). A (13)C-(13)C internuclear distance measurement from (13)CO(i) to (13)C beta(i + 3) at the periplasmic edge of the second membrane-spanning helix (TM2) of 5.1 +/- 0.2 A is consistent with the predicted alpha-helical structure and thus demonstrates an accurate long-distance rotational resonance measurement in the 120 kDa membrane-bound receptor. These measurements require a correction for the rotational resonance exchange between the multiple labels of the non-unique amino acid and the natural-abundance (13)C, which is critical to distance measurements in complex systems. A second (13)C-(13)C distance measurement between the transmembrane helices provides a high-resolution measurement of tertiary structure in the transmembrane region. The measured 5.0-5.3 A distance in the presence and absence of ligand is consistent with structural models for the transmembrane region and a proposed signaling mechanism in which ligand binding induces a 1.6 A translation of TM2. This approach can be used for additional measurements of the structure of the transmembrane region and to determine whether the ligand-induced motion is indeed propagated through the transmembrane helices.     

Size matters: non-LTR retrotransposable elements and ectopic recombination in Drosophila

The Drosophila melanogaster genome contains approximately 100 distinct families of transposable elements (TEs). In the euchromatic part of the genome, each family is present in a small number of copies (5-150 copies), with individual copies of TEs often present at very low frequencies in populations. This pattern is likely to reflect a balance between the inflow of TEs by transposition and the removal of TEs by natural selection. The nature of natural selection acting against TEs remains controversial. We provide evidence that selection against chromosome abnormalities caused by ectopic recombination limits the spread of some TEs. We also demonstrate for the first time that some TE families in the Drosophila euchromatin appear to be only marginally affected by purifying selection and contain many copies at high population frequencies. We argue that TEs in these families attain high population frequencies and even reach fixation as a result of low family-wide transposition rates leading to low TE copy numbers and consequently reduced strength of selection acting on individual TE copies. Fixation of TEs in these families should provide an upward pressure on the size of intergenic sequences counterbalancing rapid DNA loss through small deletions. Copy-number-dependent selection on TE families caused by ectopic recombination may also promote diversity among TEs in the Drosophila genome.     

Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae

Recombination plays a central role in the repair of broken chromosomes in all eukaryotes. We carried out a systematic study of mitotic recombination. Using several assays, we established the chronological sequence of events necessary to repair a single double-strand break. Once a chromosome is broken, yeast cells become immediately committed to recombinational repair. Recombination is completed within an hour and exhibits two kinetic gaps. By using this kinetic framework we also characterized the role played by several proteins in the recombinational process. In the absence of Rad52, the broken chromosome ends, both 5' and 3', are rapidly degraded. This is not due to the inability to recombine, since the 3' single-stranded DNA ends are stable in a strain lacking donor sequences. Rad57 is required for two consecutive strand exchange reactions. Surprisingly, we found that the Srs2 helicase also plays an early positive role in the recombination process.     

Induction of antitumor immunity by indomethacin

Irradiated tumor cells given, together with indomethacin, to syngeneic mice induced an antitumor response and conferred protection against a challenge of a lethal dose of murine mammary (4T1) and lung (3LL) carcinoma cells. Continuous administration of indomethacin was crucial throughout the entire period of immunization and challenge, as no protection was achieved when the drug was given during only one of these procedures. Antitumor immunity was long-lasting and, when tested in the 4T1 model, 48% of mice were resistant to a second challenge of lethal tumor cells. Tumor-free immune mice that were given indomethacin for more than 300 days remained healthy with normal white blood cell counts and normal spleen size. Cells isolated from immune mice were able to kill tumor cells in culture after in vitro activation by interleukin-2, in a manner similar to cells from naive normal control mice. In addition, the mitogenic response of their T cells was as high as that of the control naive mice. While indomethacin was able to induce antitumor immunity to 4T1 and 3LL murine carcinoma cells, both of which contain a high concentration of endogenic prostaglandin E₂ (PGE2), no such immunity was achieved to murine tumor cells with a low concentration of endogenic PGE2. These results suggest a correlation between PGE2 concentration and the ability of indomethacin to induce antitumor immunity. We therefore suggest that an immunotherapy protocol with long-term dispensation of a tolerable dose of an immunomodulator, given together with irradiated autologous tumor cells, may stimulate antitumor responses to tumors containing high concentrations of endogenic PGE2. Received: 12 August 1999 / Accepted: 21 September 1999     

Predicting and controlling the reactivity of immune cell populations against cancer

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor‐infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation‐based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti‐tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.     

Prospective Large-Scale Field Study Generates Predictive Model Identifying Major Contributors to Colony Losses

Over the last decade, unusually high losses of colonies have been reported by beekeepers across the USA. Multiple factors such as Varroa destructor, bee viruses, Nosema ceranae, weather, beekeeping practices, nutrition, and pesticides have been shown to contribute to colony losses. Here we describe a large-scale controlled trial, in which different bee pathogens, bee population, and weather conditions across winter were monitored at three locations across the USA. In order to minimize influence of various known contributing factors and their interaction, the hives in the study were not treated with antibiotics or miticides. Additionally, the hives were kept at one location and were not exposed to potential stress factors associated with migration. Our results show that a linear association between load of viruses (DWV or IAPV) in Varroa and bees is present at high Varroa infestation levels (>3 mites per 100 bees). The collection of comprehensive data allowed us to draw a predictive model of colony losses and to show that Varroa destructor, along with bee viruses, mainly DWV replication, contributes to approximately 70% of colony losses. This correlation further supports the claim that insufficient control of the virus-vectoring Varroa mite would result in increased hive loss. The predictive model also indicates that a single factor may not be sufficient to trigger colony losses, whereas a combination of stressors appears to impact hive health.