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The effect of the inhibitor of endogenous ABA synthesis fluridone on the content and distribution of endogenous ABA and IAA in the calli of ABA-deficient mutant AZ34 barley and its parental cultivar Steptoe was studied using the methods of immuno-enzymatic solid-phase assay and immunolocalization of phytohormones. It was found that by the 4th week of in vitro culture, fluridone causes a significant decrease in the ABA level in calli of both genotypes compared to the control, and the inhibitory effect of fluridone in AZ34 is more pronounced than in Steptoe. In the calli of both genotypes, a significant increase in the IAA content was revealed against the background of a decrease in the ABA content upon treatment with fluridone as compared to the control. It was concluded that ABA plays an important role in the process of embryoido-genesis in vitro.

Osmotic stress is one of the main stresses seriously affects the growth and development of plants. Hydrogen sulfide (H2S) emerges as the third gaseous signal molecule to involve in the complex network of signaling events. Phospholipase Dδ (PLDδ), as signal enzyme, responds to many biotic or abiotic stress responses. In this study, the functions and the relationship of PLDδ and H2S in stomatal closure induced by osmotic stress were explored. Using the seedlings of ecotype (WT), PLDδ deficient mutant (pldδ), L-cysteine desulfhydrase (LCD) deficient mutant (lcd) and pldδlcd double mutant as materials, the Real-time quantitative PCR (RT-qPCR) and the stomatal aperture were analyzed. Osmotic stress induced the expressions of PLDδ and LCD. The H2S content and the activities of PLD and LCD ascended in WT under osmotic stress. The phenotypes of pldδ, lcd and pldδlcd were more sensitive to osmotic stress than WT. Compared with pldδ, the stomatal of lcd showed lower sensitivity to osmotic stress, and the stomatal aperture of pldδlcd was similar to that of lcd. Simultaneous application of PA and NaHS resulted in tighter closure of stomatal than application of either PA or NaHS alone. These results suggested that osmotic stress-triggered stomatal closure requires PLDδ and H2S in A. thaliana. LCD acted downstream of PLDδ to regulate the stomatal closure induced by osmotic stress.

AML patients with TP53 mutations have extremely poor clinical outcomes. This is due primarily to limited responses to available therapies including the highly promising FDA-approved combination of Bcl-2 inhibition by venetoclax (VEN) with hypomethylating agents (DiNardo CD et al., Blood 2020), which resulted in CR/CRi rates of 70-95% and good tolerability in elderly patients (DiNardo CD et al., Lancet Oncol 2018 and Blood 2019). Apoptosis is regulated by anti- and pro-apoptotic proteins. While p53 does not directly regulate anti-apoptotic Bcl-2 proteins that are resistance factors for VEN, p53 transcriptionally up-regulates pro-apoptotic Bcl-2 proteins. Reverse phase protein array analysis of samples from newly-diagnosed AML patients found that pro-apoptotic Bax was significantly decreased in patients with TP53 mutations (Carter BZ, ASH 2019), which, as expected, diminished the effectiveness of Bcl-2 inhibition. Thus, strategies to target additional anti-apoptotic proteins, or increase pro-apoptotic proteins, are needed to enhance the efficacy of Bcl-2 inhibition in these patients. We determined protein levels of Bcl-2 family members in isogeneic Molm13 cells with TP53-knockout (KO), or with various hotspot TP53 mutations including R175H, Y220C, M237I, R248Q, R273H, and R282W. We observed markedly decreased Bax expression, to a less degree Bak decrease, and variable alterations in other Bcl-2 proteins in these cells compared to TP53-wild-type (WT) controls. We treated the aforementioned cells with VEN or the Mcl-1 inhibitor AMG 176 and found that TP53-KO or mutant cells were more resistant to both VEN and AMG 176 compared to WT controls. However, the combination of two inhibitors was highly synergistic in both settings, controls (CI = 0.2) and TP53-KO and mutant cells (CI < 0.1). To demonstrate that the decreased sensitivity to BH3 mimetics was, at least in part, mediated through Bax reduction in the TP53-mutant cells, we treated Bax knockdown (KD) Molm13 cells with VEN, AMG 176, or both. The Bax KD cells were resistant to VEN and AMG 176, while the combination of the two agents synergistically induced cell death. To establish potential clinical relevance of co-targeting Bcl-2 and Mcl-1 in TP53-mutant AML, we co-cultured cells from various TP53-mutant AML patients (n = 8) with mesenchymal stromal cells and treated them with VEN, AMG 176, or both. The combination synergistically induced cell death in both CD45 + leukemia blasts (CI values between 0.04 ± 0.04 to 0.34 ± 0.10) and CD34 + AML stem/progenitor cells (CI values between 0.07 ± 0.08 to 0.28 ± 0.14). RNA-sequencing of mononuclear and MRD cells of clinical samples (Issa G, ASH 2019) collected after induction therapy revealed that Mcl-1 expression was significantly higher in the TP53-mutated mononuclear and MRD cells compared to their WT counterparts (Fig. 1), which suggests that Mcl-1 contributes to treatment resistance and disease relapse. This further suggests that Mcl-1 inhibition should be incorporated in AML treatment, including VEN-based therapies, for patients with TP53 mutations. Finally, we treated NSG mice inoculated with isogeneic TP53-WT luciferase/GFP-labeled Molm13 and BFP-labeled TP53 R248W/R213* Molm13 cells (10:1) with VEN, AMG 176, or their combination. Only the combination treatment markedly decreased the number of GFP- and BFP-labeled cells in circulation and significantly prolonged mouse survival (median 23 d, 25 d, 24.5 d for control, VEN, AMG 176, respectively; and 45 d for VEN + AMG 176: P = 0.0007, 0.0009, and 0.0011 of combination vs. control, VEN, and AMG 176, respectively) (Fig. 2). Collectively, we demonstrate that decreased Bax contributes to resistance of TP53-mutant AML to BH3 mimetics. Mcl-1 expression positively impacts therapy resistance and disease reoccurrence in TP53-mutant AML. Thus, targeting Bcl-2 or Mcl-1 individually is insufficient and inhibition of both proteins is needed to shift cell fate from survival to death and circumvents resistance of TP53 deficient/mutant AML and AML stem/progenitor cells to BH3 mimetics. The concept warrants further clinical evaluation. Figure 1 Figure 1. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Andreeff: Medicxi: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: Research Funding; ONO Pharmaceuticals: Research Funding; Karyopharm: Research Funding; Syndax: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; Glycomimetics: Consultancy; Oxford Biomedica UK: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

The complete chloroplast genome of a novel chlorophyll-deficient mutant (clm) and its wild type (WT) in sweetpotato (Ipomoea batatas L.) was sequenced. The complete chloroplast genome of clm and WT was 161,393 bp and 161,429 bp in length, containing a large single copy (LSC) region of 87,561 bp and 87,597 bp, respectively, a small single copy (SSC) region with the same length of 30,890 bp and a pair of inverted repeat regions (IRs) with the same length of 12,052 bp. Both of them contained 132 genes including 87 protein-coding sequences, 37 tRNA, and eight rRNA. Comparing to the WT, four SNPs and three INDELs were detected and only one INDEL in the exon affecting the translation of rpoA gene. Phylogenetic analysis showed that clm and WT were closely related to Ipomoea tabascana. The complete chloroplast genome of clm and its WT will play a role in understanding the molecular mechanism of chlorophyll deficiency and developing molecular markers in sweetpotato.

Type I sourdough fermentations with Lactobacillus sanfranciscensis as predominant organism accumulate reduced glutathione through glutathione reductase (GshR) activity of L. sanfranciscensis. Reduced glutathione acts as chain terminator for gluten polymerization but is also kokumi-active and may thus enhance bread taste. This study implemented a type I model sourdough fermentations to quantitate glutathione accumulation sourdough, bread dough, and bread and to assess the effect of L. sanfranciscensis GshR on bread volume by comparison of L. sanfranciscensis and an isogenic strain devoid of GshR. L. sanfranciscensis sourdough accumulated the highest amount of reduced glutathione during proofing. Bread produced with the wild type strain had a lower volume when compared to the gshR deficient mutant. The accumulation of γ-glutamyl-cysteine was also higher in L. sanfranciscensis sourdoughs when compared to doughs fermented with the gshR mutant strain. The accumulation of reduced glutathione in L. sanfranciscensis bread did not enhance the saltiness of bread.

Lung cancer is the number 1 cause of cancer-related casualties in the world. Appropriate diagnostic markers and novel targets for lung cancer are needed. Chitooligosaccharide deacetylase homolog (YDJC) catalyzes the deacetylation of acetylated carbohydrates; however, the role of YDJC in lung cancer progression has yet to be studied. A549 lung cancer orthotopic mouse model was used for mice experiments. We found that YDJC overexpression contributes to lung cancer progression in an orthotopic mouse model. Long-term treatment (48 h) induces YDJC expression in sphingosylphosphorylcholine (SPC)-induced epithelial-mesenchymal transition (EMT). Gene silencing of YDJC (siYDJC) reduced N-cadherin expression and increased E-cadherin expression in SPC-induced EMT. Overexpression of YDJC reverses them but overexpression of the deacetylase deficient mutant YDJCD13A could not. Interestingly, overexpression of CDC16, a YDJC binding partner, suppressed EMT. ERK2 is activated in siCDC16-induced EMT. YDJC overexpression reduces expression of protein phosphatase 2A (PP2A), whereas CDC16 overexpression induces PP2A expression. YDJC overexpression induced ubiquitination of PP2A but YDJCD13A could not. CDC16 overexpression increased the ubiquitination of YDJC. These results suggest that YDJC contributes to the progression of lung cancer via enhancing EMT by inducing the ubiquitination of PP2A. Therefore, YDJC might be a new target for antitumor therapy against lung cancer.

A laboratory colony of Coleomegilla maculata (DeGeer), ye, selected for a pigmentation deficiency, was restored to near wild type cuticle coloration by adding crushed heads and wings of the red colored parental strain to the diet. While the wings and other colored portions of the cuticle re-gained the red color, the eyes of the pigmentation deficient insects were not changed from the pale mutant form. Plant derived carotenes lycopene and beta-carotene did not restore the mutant beetles to a visibly distinguishable red color. An additional pigmentation deficient mutant strain, gold, partially recovered red cuticle color when provided with diet containing pigmented insect particles. This work represents the first rescue of a color phenotype in a lady beetle.

This work aims to enhance the bioproduction of xanthan gum by screening a hyper-yield producer from the wild-type Xanthomonas campestris during a long-term continuous subculture. We reported a cell-wall deficient mutant, which performed a shift of cell morphology from rod-shaped to round-shaped. Both the yield of xanthan gum and the conversion rate of feedstock were assessed using sucrose as a carbon source with the supplement of yeast extract powder, l-glutamic acid, and other raw materials. After 96 h aerobic fermentation, the yield of xanthan gum of the mutant reached up to 32 g/L, which was 3.4 times of that of the wild-type strain. The conversion rate of feedstock in the mutant was up to 92.1%, which was 3 times of that of the wild-type (31.2%). Furthermore, pigments generated were determined and compared. As a result, the fermentation broth of the wild-type performed an OD560nm of 0.296, which was 5.8 times of that (OD560nm = 0.051) of the mutant. Microscopy analysis showed that the percentage of free-living cells in broth affected the color of the final product. Moreover, the robustness of the fermentation performance of the cell-wall deficient mutant at a pilot scale showed potential for industrial application.

To construct a prototrophic Corynebacterium glutamicum strain that efficiently produces pyruvate from glucose, the effects of inactivating RamA, a global regulator responsible for activating the oxidative tricarboxylic acid (TCA) cycle, on glucose metabolism were investigated. ΔramA showed an increased specific glucose consumption rate, decreased growth, comparable pyruvate production, higher formation of lactate and acetate, and lower accumulation of succinate and 2-oxoglutarate compared to the wild type. A significant decrease in pyruvate dehydrogenase complex activity was observed for ΔramA, indicating reduced carbon flow to the TCA cycle in ΔramA. To create an efficient pyruvate producer, the ramA gene was deleted in a strain lacking the genes involved in all known lactate- and acetate-producing pathways. The resulting mutant produced 161 mM pyruvate from 222 mM glucose, which was significantly higher than that of the parent (89.3 mM; 1.80-fold). Engineering of Corynebacterium glutamicum as a prototrophic pyruvate-producing strain: Characterization of a ramA-deficient mutant and its application for metabolic engineering

An intermuscular bone-deficient grass carp, Ctenopharyngodon idellus , mutant has been identified in an artificial gynogenetic group, in which traits encoded by recessive mutant genes can be detected because of the genomic homozygosis. Both the axial and appendicular skeletons in the mutant are developed as in the gynogenetic wild-type individuals. No growth or morphological abnormalities have been observed in the mutant. These results indicate that: (1) it is unlikely that intermuscular bone is essential for body support and normal movement of grass carp; (2) the developmental genetic regulatory mechanism of intermuscular bone differs from that of the axial and appendicular skeleton; and (3) the intermuscular bone-deficient grass carp strain can be bred using advanced biological techniques. Statistical analysis revealed that the ratio of mutant grass carp with no intermuscular bone is very low in gynogenetic grass carp groups, suggesting that many genes are involved in the development of intermuscular bones and the regulatory network is quite complex. This mutant provides unique experimental material for the identification of genes involved in intermuscular bone development.

A new super-hard rice cultivar, 'Chikushi-kona 85', which was derived from a cross between 'Fukei 2032' and 'EM129', was developed via bulk method breeding. 'Chikushi-kona 85' showed a higher content of resistant starch than the normal non-glutinous rice cultivar, 'Nishihomare', and a higher grain yield than the first super-hard rice cultivar, 'EM10'. The amylopectin chain length of 'Chikushi-kona 85' and its progenitor line 'EM129' was longer than that of 'Nishihomare', and was similar to that of 'EM10'. This suggests that the starch property of 'Chikushi-kona 85' was inherited from 'EM129', which is a mutant line deficient in a starch branching enzyme similar to 'EM10'. Genetic analysis of 'Chikushi-kona 85' crossed with 'Nishihomare' also showed that the starch property of 'Chikushi-kona 85' was regulated by a single recessive gene. Consumption of processed cookies made from 'Chikushi-kona 85' flour showed a distinctive effect in controlling blood sugar levels in comparison to the normal non-glutinous rice cultivar 'Hinohikari'. These results show that 'Chikushi-kona 85' is a novel genetic source to develop new products made of rice, which could reduce calorie intake and contribute to additional health benefits.

Under drought conditions, leaf photosynthesis is limited by the supply of CO2. Drought induces production of abscisic acid (ABA), and ABA decreases stomatal conductance (gs). Previous papers reported that the drought stress also causes the decrease in mesophyll conductance (gm). However, the relationships between ABA content and gm are unclear. We investigated the responses of gm to the leaf ABA content [(ABA)L] using an ABA-deficient mutant, aba1, and the wild type (WT) of Nicotiana plumbaginifolia. We also measured leaf water potential (ΨL) because leaf hydraulics may be related to gm. Under drought conditions, gm decreased with the increase in (ABA)L in WT, whereas both (ABA)L and gm were unchanged by the drought treatment in aba1. Exogenously applied ABA decreased gm in both WT and aba1 in a dose-dependent manner. ΨL in WT was decreased by the drought treatment to −0.7 MPa, whereas ΨL in aba1 was around −0.8 MPa even under the well-watered conditions and unchanged by the drought treatment. From these results, we conclude that the increase in (ABA)L is crucial for the decrease in gm under drought conditions. We discuss possible relationships between the decrease in gm and changes in the leaf hydraulics.

In spite of recent progress in AML therapy, the outcomes of TP53 mutated AML remain extremely poor. The FDA-approved combination of Bcl-2 inhibition by venetoclax (VEN) with hypomethylating agents is resulting in CR/CRi rates of 70-95% and good tolerability in elderly AML patients. However, patients with TP53 mutations achieve lower response rates (CR/CRi 47%) (DiNardo CD et al., Lancet Oncol 2018; DiNardo CD et al., Blood 2019) and invariably relapse. Combined Bcl-2 inhibition and p53 activation is synthetic lethal in TP53 wild-type AML in part through targeting Mcl-1 (Pan R et al., Cancer Cell 2016). In TP53 deficient/mutant AML, direct targeting of Mcl-1 may partially compensate for the TP53 defect. We therefore postulate that combined inhibition of Mcl-1 and Bcl-2 effectively induces apoptosis in TP53 deficient/mutant AML cells. Reverse phase protein array analysis of a large cohort of newly diagnosed AML patients (n=511) enabled us to stratify patients into various prognostic groups based on p53 pathway protein expression, which included 8 core proteins: TP53, TP53pS15, MDM2, MDM4, TRIM24, SFN, IRS1.pS1101, and YWHAZ. The group with p53 pathway dysfunction, defined by high p53 protein levels, is characterized by poor outcomes (Quintas-Cardama A et al., Leukemia 2017). This group, encompassing both TP53 wild-type and TP53 mutations, had significantly lower expression of Bax (p=0.0007), the chief executioner of intrinsic apoptosis. A survey of Bcl-2 family proteins in TP53 wild-type and mutant AML showed that only Bax was significantly lower in patients with TP53 mutations (p=0.0498). We next investigated the roles of p53 in response to BH3 mimetics in TP53 wild-type and knockdown (KD) OCI-AML3 cells and in TP53 wild-type and mutant Molm13 cells, generated by long-term exposure of TP53 wild-type Molm13 cells to RG7388 (idasanutlin). Western blot analysis showed that Bax protein was consistently decreased in both TP53 KD and mutant cell lines compared to their respective controls. p53 KD OCI-AML3 and TP53 mutant Molm13 cells exhibited decreased sensitivity not only to VEN, but also to Mcl-1 inhibitor AZD5991 compared to OCI-AML3 vector control and Molm13 parental cells, respectively. To investigate if combined inhibition of Bcl-2 and Mcl-1 could counter-balance the loss of TP53 activity, p53 KD and TP53 mutated AML cells were treated with VEN and AZD5991: like in their respective control cells, the combination synergistically induced cell death in these cells (Fig. 1). The EC50 levels of VEN required for the described synergism in OCI-AML3 cells can easily be reached/exceeded clinically. The combination was also synergistic in leukemia cell lines lacking wild-type TP53 such as KG-1 and U937. Importantly, the combined inhibition was more effective than each single agent in primary AML cells and stem/progenitor cells lacking wild-type TP53 due to deletion of chromosome 17 or mutations in TP53 gene. Conclusion: AML cells deficient in functional TP53 have decreased Bax protein expression, increased apoptotic threshold and are more resistant to individual BH3 mimetics. Combined inhibition of Bcl-2 and Mcl-1 is highly synergistic in p53 deficient/mutant AML. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Cidado:AstraZeneca: Employment. Drew:AstraZeneca: Employment. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy.

Background Rice is the only crop that germinates and elongates the coleoptile under complete submergence. It has been shown that alcohol dehydrogenase 1 (ADH1)-deficient mutant of rice with reduced alcohol dehydrogenase activity (rad) and reduced ATP level, is viable with much reduced coleoptile elongation under such condition. To understand the altered transcriptional regulatory mechanism of this mutant, we aimed to establish possible relationships between gene expression and cis-regulatory information content. Findings We performed promoter analysis of the publicly available differentially expressed genes in ADH1 mutant. Our results revealed that a crosstalk between a number of key transcription factors (TFs) and different phytohormones altered transcriptional regulation leading to the survival of the mutant. Amongst the key TFs identified, we suggest potential involvement of MYB, bZIP, ARF and ERF as transcriptional activators and WRKY, ABI4 and MYC as transcriptional repressors of coleoptile elongation to maintain metabolite levels for the cell viability. Out of the repressors, WRKY TF is most likely playing a major role in the alteration of the physiological implications associated with the cell survival. Conclusions Overall, our analysis provides a possible transcriptional regulatory mechanism underlying the survival of the rad mutant under complete submergence in an energy crisis condition and develops hypotheses for further experimental validation. Electronic supplementary material The online version of this article (doi:10.1186/s12284-016-0124-3) contains supplementary material, which is available to authorized users.

The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.

Acyl-acyl carrier protein (ACP) thioesterases are enzymes that control the termination of intraplastidial fatty acid synthesis by hydrolyzing the acyl-ACP complexes. Among the different thioesterase gene families found in plants, the FatA-type fulfills a fundamental role in the export of the C18 fatty acid moieties that will be used to synthesize most plant glycerolipids. A reverse genomic approach has been used to study the FatA thioesterase in seed oil accumulation by screening different mutant collections of Arabidopsis thaliana for FatA knockouts. Two mutants were identified with T-DNA insertions in the promoter region of each of the two copies of FatA present in the Arabidopsis genome, from which a double FatA Arabidopsis mutant was made. The expression of both forms of FatA thioesterases was reduced in this double mutant (fata1 fata2), as was FatA activity. This decrease did not cause any evident morphological changes in the mutant plants, although the partial reduction of this activity affected the oil content and fatty acid composition of the Arabidopsis seeds. Thus, dry mutant seeds had less triacylglycerol content, while other neutral lipids like diacylglycerols were not affected. Furthermore, the metabolic flow of the different glycerolipid species into seed oil in the developing seeds was reduced at different stages of seed formation in the fata1 fata2 line. This diminished metabolic flow induced increases in the proportion of linolenic and erucic fatty acids in the seed oil, in a similar way as previously reported for the wri1 Arabidopsis mutant that accumulates oil poorly. The similarities between these two mutants and the origin of their phenotype are discussed in function of the results. © 2011 Springer-Verlag., This work was supported by the Spanish MICINN and FEDER, Project AGL2008-01086/ALI.

Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

We report here the complete genome sequence of the Aneurinibacillus migulanus E1 mutant deficient in gramicidin S (GS) and d -phenylalanyl- l -propyl diketopiperazine (DKP) formation. The genome consists of a circular chromosome (6,301,904 bp, 43.20% G+C content) without any plasmid. The complete genome sequence enables further investigation of the biosynthetic mechanism and the biological function of gramicidin S.

Pseudomonas aeruginosa produces rhamnolipids which are tenso-active compounds with potential industrial and environmental applications. There are two main types of rhamnolipids produced in liquid cultures, rhamnosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (mono-rhamnolipid) and rhamnosyl-rhamnosyl-beta-hydroxydecanoyl-beta-hydroxyd ecanoate (di-rhamnolipid). In this work we report the selective isolation of a rhamnolipid deficient mutant (IBT8), which does not accumulate mono-rhamnolipid while still producing di-rhamnolipid. IBT8 was selected after random mutagenesis with Tn501; yet, its mono-rhamnolipid deficiency was found associated neither with its Tn501 insertion nor with a possible alteration in the rhlABRI genes for rhamnosyl-transferase 1 synthesis. Different possibilities to explain IBT8 phenotype are discussed.

Potato leafroll virus (PLRV), gênero Polerovirus, família Luteoviridae, é transmitido por afídeos de um modo persistente e circulativo. Membros da família Luteoviridae associam-se a um homólogo de GroEL produzido pelo endosimbionte primário (Buchnera sp.) de afídeos para evitar a degradação na hemolinfa. Partículas purificadas de luteovirus contêm dois tipos de proteínas: a capa protéica (CP) de ~22 kDa e um componente "capsidial" de 54 kDa, o qual é uma forma truncada de uma proteína de "transleitura" a partir do códon de terminação do gene da CP. O domínio de transleitura (RTD) contém determinantes responsáveis pela transmissão do vírus. Um clone de cDNA infeccioso do PLRV e um mutante deletério da RTD foram usados para analisar as interações entre esse luteovirus e seu afídeo vetor Myzus persicae. As partículas mutantes do PLRV, deficientes da proteína RTD inteira, não foram transmissíveis por M. persicae e não se ligaram a Buchnera GroEL. Adicionalmente, esse mutante foi menos persistente na hemolinfa do afídeo do que o vírus selvagem.

The mutant strain (ha) of medaka (Oryzias latipes) lack utricular otoliths as fry, and some never form otoliths for life. The cross (F1 generation) between the strain having good eyesight and another strain having ordinary eyesight augmented visual acuity of the F1 generation. Crossing the good eyesight strain and ha mutant produced fish having good eyesight and less sensitivity to gravity in the F2 population. Their tolerance to microgravity was tested by parabolic flight using an airplane. The fish exhibited less looping and no differences in degree of looping between light and dark conditions, suggesting that loss of eyesight (in darkness) is not a direct cause for looping behavior in microgravity. The ha embryos could not form utricular otoliths. They did form saccular otoliths, but with a delay. Fry of the mutant fish lacking the utricular otoliths are highly dependent on light upon hatching and exhibit a perfect dorsal-light response (DLR). As they grow, they eventually shift from being light-dependent to being gravity-dependent. Continuous treatment of the fry with altered light direction suppressed this shift to gravity dependence. Being less dependent on gravity, these fish can serve as models in studying the differences expected for the vestibular system of fish reared in microgravity. When these fish were exposed to microgravity (parabolic flights) of an airplane, they spent far less time looping than fish reared in an ordinary light regimen.

Kinetochores must continuously associate with dynamic microtubule plus ends, as they oscillate along the mitotic spindle. The molecular basis for the kinetochore to track microtubule plus ends remains unresolved. In a recent study, we have shown an essential role of the formin mDia3 in stable kinetochore microtubule attachment and metaphase chromosome alignment. This function is attributable to EB1-binding by mDia3, for replacing endogenous mDia3 with an EB1-binding deficient mutant results in chromosome misalignment. EB1 specifically targets to attached, antipoleward kinetochores with polymerizing microtubules during chromosome oscillation. Therefore, we speculate that the mDai3-EB1-APC complex formation may relay EB1 microtubule plus end-tracking activity to the kinetochore.

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Ycf1 and Acr3 are transporters that have been previously shown to protect Saccharomyces cerevisiae cells from the toxic effects of arsenite. Ycf1 and Acr3 are positively regulated by distinct, but related bZIP transcriptional activators, Yap1 and Yap8, respectively. In this study, we show that overexpression of Yap1 complemented the arsenite hypersensitivity of the ycf1 null mutant, but only if the ACR3 gene is functional. We further show that the expression of either an ACR3-lacZ promoter fusion reporter or the endogenous ACR3 gene was stimulated by the overproduction of Yap1 upon exposure to arsenite. These data suggest that Yap1 confers arsenite resistance to the ycf1 null mutant by activating expression of the Yap8-dependent target gene, ACR3. Our data also show Yap8-dependent ACR3-lacZ expression was greatly stimulated by arsenite in a dose-dependent manner in the parental strain. However, overproduction of Yap1 in the parental strain severely limited dose-dependent activation of the reporter by arsenite. We conclude that Yap1 may compete with Yap8 for binding to the ACR3 promoter, but is unable to act as a potent activator.Key words: arsenite, ABC transporters, AP-1 factors, overproduction, element, yeast.

Lack of triose phosphate isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae ΔTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, andPseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.Key words: Gluconobacter orphans, recA gene, DNA repair, recA mutant, SOS box.