Differential hypoxia response of hsp-16 genes in the nematode

Small heat shock proteins are induced by various stresses. We here report the differential hypoxia responses of the hsp-16 genes in the nematode. The hsp-16.1 and hsp-16.2 genes in Caenorhabditis elegans responded to hypoxia, while hsp-16.41 and hsp-16.48, which share the promoter regions with hsp-16.1 and hsp-16.2, respectively, did not. For comparative genomic analysis, we identified ten hsp-16 genes in the nematode C.briggsae from the genome database. The comparison of the promoter sequences revealed a new conserved sequence block, CAC(A/T)CT, that was required for the orientation-dependent hypoxia response, but not for other stress responses such as heat or ethanol. We propose a working model for the orientation-dependent promoter usage between two genes sharing the promoter region. We also discuss a possible application of the hypoxia-inducible promoter for conditional gene expression.     

Suppression of heat-induced HSP-70 by simultaneous exposure to 50 mT magnetic field

Effect of extremely low frequency magnetic field (ELFMF) at 50 mT and 60 Hz on heat-induced expression of heat shock protein 70 (hsp-70) was examined in HL60RG cells. No increase in hsp-70 production was observed in the cells after exposure to 50 mT ELFMF alone. Simultaneous exposure to 50 mT ELFMF in combination with mild heat at 42 and 40 degrees C suppressed heat-induced hsp-70 expression. The suppression of hsp-70 occurred when cells were simultaneously exposed to both for longer periods of more than 5 h. However, the suppression of hsp-70 was not observed at a magnetic density of 5 and 0.5 mT. This result suggests that exposure to 50 mT ELFMF may act on a protection against the concomitant mild heat stress in HL60RG cells.     

Continuous wave and simulated GSM exposure at 1.8 W/kg and 1.8 GHz do not induce hsp16-1 heat-shock gene expression in Caenorhabditis elegans

Recent data suggest that there might be a subtle thermal explanation for the apparent induction by radiofrequency (RF) radiation of transgene expression from a small heat-shock protein (hsp16-1) promoter in the nematode, Caenorhabditis elegans. The RF fields used in the C. elegans study were much weaker (SAR 5-40 mW kg(-1)) than those routinely tested in many other published studies (SAR approximately 2 W kg(-1)). To resolve this disparity, we have exposed the same transgenic hsp16-1::lacZ strain of C. elegans (PC72) to higher intensity RF fields (1.8 GHz; SAR approximately 1.8 W kg(-1)). For both continuous wave (CW) and Talk-pulsed RF exposures (2.5 h at 25 degrees C), there was no indication that RF exposure could induce reporter expression above sham control levels. Thus, at much higher induced RF field strength (close to the maximum permitted exposure from a mobile telephone handset), this particular nematode heat-shock gene is not up-regulated. However, under conditions where background reporter expression was moderately elevated in the sham controls (perhaps as a result of some unknown co-stressor), we found some evidence that reporter expression may be reduced by approximately 15% following exposure to either Talk-pulsed or CW RF fields.     

Direct observation of stress response in Caenorhabditis elegans using a reporter transgene

Transgenic Caenorhabditis elegans expressing jellyfish Green Fluorescent Protein under the control of the promoter for the inducible small heat shock protein gene hsp-16-2 have been constructed. Transgene expression parallels that of the endogenous hsp-16 gene, and, therefore, allows direct visualization, localization, and quantitation of hsp-16 expression in living animals. In addition to the expected upregulation by heat shock, we show that a variety of stresses, including exposure to superoxide-generating redox-cycling quinones and the expression of the human beta amyloid peptide, specifically induce the reporter transgene. The quinone induction is suppressed by coincubation with L-ascorbate. The ability to directly observe the stress response in living animals significantly simplifies the identification of both exogenous treatments and genetic alterations that modulate stress response, and possibly life span, in C. elegans.     

Identification of a heat-shock pseudogene from Caenorhabditis elegans

While characterizing the hsp70 gene family from Caenorhabditis elegans we encountered an unusual member of this family. Sequence data reveal that the hsp-2ps gene is a pseudogene of the constitutively expressed, heat-inducible hsp-1 gene. Two stop codons generated near the 5' end of the sequence as well as several frameshift mutations and a large internal deletion confirm the identification of hsp-2ps as a pseudogene. The nucleotide substitution rate of the third codon position was twice that of the first and second codon positions, suggesting that the hsp-2ps gene was nonfunctional since the time of the duplication event. The hsp-2ps gene duplicates a region of the hsp-1 gene that lies exclusively within the transcribed region and retains the introns. We feel that the hsp-2ps gene was produced by a transpositional duplication event, which occurred approximately 8.5 million years ago.     

Identification of human hsp-70 expression in stably transfected rodent cells by isoelectrofocus immunoblots.

The major heat-shock protein, hsp-70, is synthesized by cells from a wide variety of organisms in response to heat shock or other stresses. It is assumed that hsp-70 may have an important thermal protective function. To test this hypothesis directly, we have transfected rat fibroblast cells with appropriate expression plasmids containing a cloned human hsp-70 gene. Stable transfectants expressing the human hsp-70 gene product were identified by Western blot analysis. During the course of selecting successful transfectants, we found that when standard methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunostaining were employed with the monoclonal antibody raised against the human hsp-70 antigen, we were unable to differentiate the human hsp-70 from the heat-inducible rat hsp-70. This was because the monoclonal antibody cross-reacts with the human and rat proteins, which have the same mobility in SDS-PAGE, and it is difficult to determine which protein is expressed. To improve the resolution of the Western blot technique, we performed additional immunoblot analysis of cellular proteins separated by slab gel isoelectrofocusing. Our study shows that the isoelectrofocusing technique, when combined with antibodies against hsp-70, gave a better resolution for the separation of exogenous human hsp-70 and the endogenous rat hsp-70 than the commonly used SDS-PAGE Western blot analysis. It provides a rapid and specific method to identify positive colonies that express the human hsp-70 gene in transfected rodent cells.     

Extremely low frequency electromagnetic field exposure promotes differentiation of pituitary corticotrope-derived AtT20 D16V cells

The pituitary corticotrope-derived AtT20 D16V cell line responds to nerve growth factor (NGF) by extending neurite-like processes and differentiating into neurosecretory-like cells. The aim of this work is the study of the effect of extremely low frequency electromagnetic fields (ELF-EMF) at a frequency of 50 Hz on these differentiation activities. To establish whether exposure to the field could influence the molecular biology of the cells, they were exposed to a magnetic flux density of 2 milli-Tesla (mT). Intracellular calcium ([Ca2+]i) and intracellular pH (pHi) were monitored in single exposed AtT20 D16V cells using fluorophores Indo-1 and SNARF for [Ca2+]i and pHi, respectively. Single-cell fluorescence microscopy showed a statistically significant increase in [Ca2+]i followed by a drop in pHi in exposed cells. Both scanning electron microscopy (SEM) and transmission microscopy of exposed AtT20 D16V cells show morphological changes in plasma membrane compared to non-exposed cells; this modification was accompanied by a rearrangement in actin filament distribution and the emergence of properties typical of peptidergic neuronal cells-the appearance of secretory-like granules in the cytosol and the increase of synaptophysin in synaptic vesicles, changes typical of neurosecretory-like cells. Using a monoclonal antibody toward the neurofilament protein NF-200 gave additional evidence that exposed cells were in an early stage of differentiation compared to control. Pre-treatment with 0.3 microM nifedipine, which specifically blocks L-type Ca2+ channels, prevented NF-200 expression in AtT20 D16V exposed cells. The above findings demonstrate that exposure to 50 Hz ELF-EMF is responsible for the premature differentiation in AtT20 D 16 V cells.     

Quantification of Expression of Staphylococcus epidermidis Housekeeping Genes with Taqman Quantitative PCR during In Vitro Growth and under Different Conditions

The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene; hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10 degrees C heat shock (37 to 47 degrees C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for Escherichia coli that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).     

A novel synthetic peptide vector system for optimal gene delivery to bone marrow stromal cells.

A 23-amino acid, bifunctional, integrin-targeted synthetic peptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). The peptide (K)(16)GRGDSPC consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 7-amino acid integrin-binding domain at the carboxyl terminal. PcDNA3-EGFP plasmids were transfected into BMSCs by (K)(16)GRGDSPC and the positive cells gave out a bright green fluorescence. High levels of gene delivery of pcDNA3-TGF-beta1 plasmids were obtained with 2 to 4 microg/ml DNA concentration, with (K)(16)GRGDSPC at an optimal peptide: DNA w/w ratio of 3:1, with a required exposure time of more than 4 h but shorter than 24 h for BMSC exposure to the peptide/DNA complexes with completely absent serum in the initial stage; with 100 microM chloroquine and at least 8 h exposure for BMSC exposure to chloroquine; with a fusogenic peptide at an optimal (K)(16)GRGDSPC/DNA/fusogenic peptide w/w ratio of 3:1:5; and with Lipofectamine 2000 at an optimal (K)(16)GRGDSPC/DNA/Lipofectamine 2000 w/w ratio of 3:1:2 at a constant DNA concentration of 2 microg/ml. Chloroquine, the fusogenic peptide and Lipofectamine 2000 all significantly promoted gene delivery, but chloroquine was more effective than the fusogenic peptide and had obvious synergistic effects with Lipofectamine 2000. Under optimal conditions, TGF-beta1 gene was transfected into BMSCs without observable toxicity, and the stable expression was examined by RT-PCR and Western blot analysis. The stable transgenic cells showed obvious bands. This novel synthetic peptide, providing a new way for the use of polylysine and RGD motif in DNA vector system, is potentially well suited to ex vivo gene delivery to BMSCs for experimental and clinical applications in the field of bone tissue engineering.     

Heritable transgenesis of Parastrongyloides trichosuri: a nematode parasite of mammals

Germline transformation of a parasitic nematode of mammals has proven to be an elusive goal. We report here the heritable germline transformation of Parastrongyloides trichosuri, a nematode parasite whose natural hosts are Australian possums of the genus Trichosurus. This parasite can undergo multiple free-living life cycles and these replicative cycles can be maintained indefinitely in the laboratory. Transformation was achieved by microinjection of DNA into the ovary syncytium of either free-living or parasitic adult females. By selecting for the transgenic progeny of successive free-living life cycles, it was possible to establish and maintain transgenic lines. All three transgenic lines tested were shown capable of establishing patent infections in possums and to transmit the functional transgene to their progeny. The transgene, driven by the Pt hsp-1 promoter, was constitutively expressed in intestinal cells at all stages of both parasitic and free-living life cycles, although gene silencing appears to occur in some transgenic progeny. This is the first report of heritable transgenesis in a parasitic nematode of a mammal and we discuss a variety of previously inaccessible experimental avenues that will now be possible with this powerful model system.     

Herpes simplex virus transcriptional activator VP16 is detrimental to preimplantation development in mice

The herpes simplex virus transactivator protein VP16 is frequently used to regulate gene expression in several experimental systems, including transgenic mice. It has been suggested that high levels of VP16 expression in mice may be lethal. In order to systematically address this issue, we linked the VP16 gene to promoters that are active early and in a variety of tissues throughout development, such as the human beta-actin promoter or the rat nestin gene enhancer. VP16 expression was assayed using a LacZ reporter gene linked to a VP16-responsive immediate early gene promoter. We show here that expression of VP16 at high levels is detrimental to pre-implantation development. By culturing embryos in vitro, we demonstrate that this effect is exerted at the transition from the 2-cell to the 4-cell stage, reducing survival to the blastocyst stage dramatically. On the other hand, transgenic mice expressing VP16 transgenes at postimplantation stages are viable. These results suggest a differential sensitivity to VP16 expression in different cell types and stages of development. The reduction of embryo survival by VP16 implicates herpes virus infection as a potential cause of infertility.     

Ubiquitin-like protein 5 positively regulates chaperone gene expression in the mitochondrial unfolded protein response

Perturbation of the protein-folding environment in the mitochondrial matrix selectively upregulates the expression of nuclear genes encoding mitochondrial chaperones. To identify components of the signal transduction pathway(s) mediating this mitochondrial unfolded protein response (UPR(mt)), we first isolated a temperature-sensitive mutation (zc32) that conditionally activates the UPR(mt) in C. elegans and subsequently searched for suppressors by systematic inactivation of genes. RNAi of ubl-5, a gene encoding a ubiquitin-like protein, suppresses activation of the UPR(mt) markers hsp-60::gfp and hsp-6::gfp by the zc32 mutation and by other manipulations that promote mitochondrial protein misfolding. ubl-5 (RNAi) inhibits the induction of endogenous mitochondrial chaperone encoding genes hsp-60 and hsp-6 and compromises the ability of animals to cope with mitochondrial stress. Mitochondrial morphology and assembly of multi-subunit mitochondrial complexes of biotinylated proteins are also perturbed in ubl-5(RNAi) worms, indicating that UBL-5 also counteracts physiological levels of mitochondrial stress. Induction of mitochondrial stress promotes accumulation of GFP-tagged UBL-5 in nuclei of transgenic worms, suggesting that UBL-5 effects a nuclear step required for mounting a response to the threat of mitochondrial protein misfolding.     

转几丁质酶和葡聚糖酶双价基因棉花对土壤细菌种群多样性的影响

以转几丁质酶和葡聚糖酶双价基因棉花为研究对象,非转基因受体棉花为对照,通过比较可培养细菌数量和基于16S rRNA克隆文库细菌种群分析,评价外源双价基因的导入在苗期、蕾期、花铃期和吐絮期对棉花根际细菌群落多样性的影响。结果表明,可培养细菌的数量不受外源双价基因的影响,随着棉花生育期的交替而变化,以代谢旺盛的花铃期最多。构建的转基因和非转基因不同生育期根际土壤细菌16S rRNA文库容量为2400个克隆,涵盖了细菌的283个属。其中,Acidobacterium是最大优势类群,共包括624个克隆,其次为未知细菌种群和Flavisolibacter。比较转基因和非转基因棉花根际土壤细菌的种群结构,结果显示,同一生育期内前者种群的多样性显著低于后者,二者的共有类群随着生长发育的进行而增多。研究结果说明几丁质酶基因和葡聚糖酶基因对棉花根际细菌种群多样性有着不同程度的削减作用,但是随着种植时间的延长,该差异呈现逐渐缩小的趋势。     

The Embryonically Active Gene, Unkempt, of Drosophila Encodes a Cys(3)his Finger Protein

The unkempt gene of Drosophila encodes a set of embryonic RNAs, which are abundant during early stages of embryogenesis and are present ubiquitously in most somatic tissues from the syncytial embryo through stage 15 of embryogenesis. Expression of unkempt RNAs becomes restricted predominantly to the central nervous system in stages 16 and early 17. Analysis of cDNAs from this locus reveals the presence of five Cys3His fingers in the protein product. Isolation and analysis of mutations affecting the unkempt gene, including complete deletions of this gene, indicate that there is no zygotic requirement for unkempt during embryogenesis, presumably due to the contribution of maternally supplied RNA, although the gene is essential during post-embryonic development.     

除草剂草铵膦对转基因抗草铵膦棉花生长发育的影响

【背景】草铵膦是继草甘膦之后又一种高效、广谱、低毒的非选择性除草剂,有望成为我国转基因抗除草剂棉花的主要靶标之一。目前,在抗草铵膦棉花的不同生育期使用草铵膦是否影响棉花生长发育尚不清楚。【方法】分别在抗草铵膦棉花子叶期、3~4片真叶期、现蕾期、盛花期喷施草铵膦,单次施药量分别为900和1800 g·hm-2,每个处理小区面积25.6 m2。分析草铵膦对花粉育性、棉花生长、产量及纤维品质的影响。【结果】喷施草铵膦对棉花盛花期的花粉活力、株高、茎直径、产量因子、纤维品质等各项参数均无显著性影响,在子叶期使用1800 g·hm-2的高剂量浓度处理时,籽指水平显著低于空白对照处理。【结论与意义】转基因抗草铵膦棉花对900、1800g·hm-2的草铵膦具有较好的耐受性。本研究为转基因抗草铵膦棉花的环境安全性评价提供了依据。