In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant acetohydroxyacid synthase genes

Summary Genes coding for the enzyme acetohydroxyacid synthase, often referred to as acetolactate synthase (AHAS, ALS; EC 4.1.3.18), from wild type Arabidopsis thaliana and a sulfonylurea-resistant mutant line GH50 (csrl-1; Haughn et al. 1988) were introduced in Nicotiana tabacum. Both genes were expressed at high levels with the 35S promoter. The csrl-1 gene conferred high levels of resistance to chlorsulfuron whereas the wild type gene did not. As selectable markers, chimaeric AHAS genes yielded transgenic plants on chlorsulfuron but at much lower efficiencies than with a chimaeric neomycin phosphotransferase gene on kanamycin (Sanders et al. 1987). Shoot differentiation from leaf discs was delayed on chlorsulfuron by 4–6 weeks. This study indicated a role for mutant AHAS genes in the genetic manipulation of herbicide resistance in transgenic plants but as selectable markers for plant cells undergoing differentiation no advantage over other genes was perceived.     

Effects of ozone on wild type and transgenic tobacco

Tocopherol cyclase (TC, encoded by gene VTE1) catalyzes the penultimate step of tocopherol synthesis. In this study we used wild type and transgenic tobacco plants overexpressing VTE1 from Arabidopsis to examine the role of tocopherol in ozone sensitivity. Wild type plants responded to an 4-h exposure to 300 nmol mol⁻¹ ozone by severe leaf necrosis while the transgenic lines exhibited limited injury. Compared with the wild type, VTE1-overexpressing plants had lower increase in hydrogen peroxide, malondialdehyde contents and ion leakage, and lower decrease of net photosynthetic rate 48 h following the ozone exposure. Transgenic plants also better maintained the structural integrity of the photosynthetic apparatus.     

Reelin in the extracellular matrix and dendritic spines of the cortex and hippocampus: a comparison between wild type and heterozygous reeler mice by immunoelectron microscopy

Reelin is a glycoprotein (～400 kDa) secreted by GABAergic neurons into the extracellular matrix of the neocortex and hippocampus as well as other areas of adult rodent and nonhuman primate brains. Recent findings indicate that the heterozygote reeler mouse (haploinsufficient for the reeler gene) shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder with mania. These include (1) a downregulation of both reelin mRNA and the translated proteins, (2) a decrease in the number of dendritic spines in cortical and hippocampal neurons, (3) a concomitant increase in the packing density of cortical pyramidal neurons, and (4) an age-dependent decrease in prepulse inhibition of startle. Interestingly, the heterozygous reeler mouse does not exhibit the unstable gait or the neuroanatomy characteristic of the null mutant reeler mouse. Immunocytochemical studies of the expression of reelin in mice have been primarily limited to light microscopy. In this study we present new immunoelectron microscopy data that delineates the subcellular localization of reelin in the cortex and hippocampus of the wild-type mouse, and compares these results to reelin expression in the heterozygous reeler mouse. In discontinuous areas of cortical layers I and II and the inner blade area of the dentate gyrus of the wild type mouse, extracellular reelin is associated with dendrites and dendritic spine postsynaptic specializations. Similar associations have been detected in the CA1 stratum oriens and other areas of the hippocampus. In the hippocampus, reelin expression is more expansive and more widespread than in cortical layers I and II. In contrast, extracellular reelin immunoreactivity is greatly diminished in all areas examined in the heterozygous reeler mouse. However, some cell bodies of GABAergic neurons in the cortex and hippocampus demonstrate an increased accumulation of reelin in the Golgi and endoplasmic reticulum. We suggest that in the heterozygous reeler mouse a downregulation of reelin biosynthesis results in a decreased rate of secretion into the extracellular space. This inhibits dendritic spine maturation and plasticity and leads to dissociation of dendritic postsynaptic density integrity and atrophy of spines. We speculate that the haploinsufficient reeler mouse may provide a model for future studies of the role of reelin, as it may be related to psychosis vulnerability.     

球孢白僵菌野生及转基因菌株的退化

球孢白僵菌在人工培养基上继代培养容易发生退化变异,表现为菌丝徒长以及产孢量减少和毒力的逐渐下降,给真菌杀虫剂的生产和使用造成较大的损失.没有丢失被插入的外源毒力基因的工程株同样出现退化.培养基成分和含水量以及培养温度和光照等环境因子皆可不同程度地诱发退化的发生,但退化主要由其遗传机制决定,包括线粒体基因突变、异核现象、准性生殖、核基因突变以及有性重组等;菌落局变实质上也是菌种老化的征兆.真菌杀虫剂生产中防止退化的对策除减少传代次数外,还要根据具体菌株确定适宜的环境控制细则.     

Induction of drug metabolism-related enzymes by methylcholanthrene and phenobarbital in transgenic mice carrying human prototype c-Ha-ras gene and their wild type littermates.

Transgenic mice hemizygously carrying human c-Ha-ras proto-oncogene, Tg-rasH2 show very sensitive and facilitated carcinogenicity to various carcinogens. In this study, activities of certain enzymes related to drug metabolism and energy metabolism were measured in microsome and cytosol fractions of livers of Tg-rasH2 mice and their wild type littermates with both sexes treated with 3-methylcholanthrene (MC) and phenobarbital (PB). Aminopyrine N-demethylase activities increased significantly in livers of all mice treated with PB. MC and PB treatments induced significant increases in activities of UDP-glucuronosyltransferase and S-adenosyl homocysteinase compared to those in the non-treated groups in microsome fractions from all mice. In cytosol fractions of livers of all mice, glutathione S-transferase activity was significantly induced in the PB treated groups. There were no significant differences in activities of lactate dehydrogenase, glucose 6-phosphate dehydrogenase, pyruvate kinase and glucose 6-phosphatase related to energy metabolism in livers and kidneys among all mice. Tg-rasH2 mice showed stable activities of enzymes related to drug detoxication and energy metabolism similar to those of non-transgenic mice. These results suggest that the human c-Ha-ras transgene may not affect drug metabolism-related enzymes, and the facilitated carcinogenic response in the Tg-rasH2 mouse is not due to these enzymatic disorders.     

Characterization of loricrin regulation in vitro and in transgenic mice

We have previously shown that the promoter of a 6.5 kb mouse loricrin clone contains a functional AP-1 element and directs tissue-specific, but not differentiation-specific, expression. We now report the isolation of a 14-kb genomic clone containing an additional 7 kb of genomic sequence. The additional sequences limit expression of a reporter construct to differentiated keratinocytes in culture. The expression of the 6.5-kb and 14-kb loricrin constructs were also analyzed in transgenic mice. Significantly, loricrin was found in all layers of the epidermis of the 6.5-kb transgenics, including basal and spinous cells. The expression of the 14-kb clone was indistinguishable from that of the endogenous gene, confirming that the additional sequences contain negative regulatory elements that restrict loricrin expression to the granular layer in vivo. In addition, we show the AP-1 element localized in the loricrin proximal promoter is necessary but not sufficient for expression of the loricrin gene in vivo in transgenic mice. Finally, to gain further insight into how AP-1 family members regulate expression of the loricrin gene, we co-transfected the loricrin reporter constructs with expression plasmids for various fos and jun family members and demonstrated that c-Fos/Jun-B heterodimers could mimic the differentiation-specific induction of loricrin.     

Unique lipoproteins secreted by primary astrocytes from wild type, apoE (-/-), and human apoE transgenic mice.

Composition of central nervous system lipoproteins affects the metabolism of lipoprotein constituents within the brain. The epsilon4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease via an unknown mechanism(s). As glia are the primary central nervous system cell type that synthesize apoE, we characterized lipoproteins secreted by astrocytes from wild type (WT), apoE (-/-), and apoE transgenic mice expressing human apoE3 or apoE4 in a mouse apoE (-/-) background. Nondenaturing size exclusion chromatography demonstrates that WT, apoE3, and apoE4 astrocytes secrete particles the size of plasma high density lipoprotein (HDL) composed of phospholipid, free cholesterol, and protein, primarily apoE and apoJ. However, the lipid:apoE ratio of particles containing human apoE is significantly lower than WT. ApoE localizes across HDL-like particle sizes. ApoJ localizes to the smallest HDL-like particles. ApoE (-/-) astrocytes secrete little phospholipid or free cholesterol despite comparable apoJ expression, suggesting that apoE is required for normal secretion of astrocyte lipoproteins. Further, particles were not detected in apoE (-/-) samples by electron microscopy. Nondenaturing immunoprecipitation experiments indicate that apoE and apoJ reside predominantly on distinct particles. These studies suggest that apoE expression influences the unique structure of astrocyte lipoproteins, a process further modified by apoE species.     

Bacterial community in the rhizosphere and rhizoplane of wild type and transgenic eucalyptus

The rhizosphere is a niche exploited by a wide variety of bacteria. The expression of heterologous genes by plants might become a factor affecting the structure of bacterial communities in the rhizosphere. In a greenhouse experiment, the bacterial community associated to transgenic eucalyptus, carrying the Lhcb1-2 genes from pea (responsible for a higher photosynthetic capacity), was evaluated. The culturable bacterial community associated to transgenic and wild type plants were not different in density, and the Amplified Ribosomal DNA Restriction Analysis (ARDRA) typing of 124 strains revealed dominant ribotypes representing the bacterial orders Burkholderiales, Rhizobiales, and Actinomycetales, the families Xanthomonadaceae, and Bacillaceae, and the genus Mycobacterium. Principal Component Analysis based on the fingerprints obtained by culture-independent Denaturing Gradient Gel Electrophoresis analysis revealed that Alphaproteobacteria, Betaproteobacteria and Actinobacteria communities responded differently to plant genotypes. Similar effects for the cultivation of transgenic eucalyptus to those observed when two genotype-distinct wild type plants are compared.     

Development and morphological features of biofilms formed by transgenic and wild type strains of Bacillus subtilis

The study addressed the ability of the transgenic strain (TM) B. subtilis 2335/pBMB105 (Km^rInf⁺) to form biofilms on the surface of liquid media of various compositions, inoculated with vegetative cells and spores. The morphological features of these biofilms do not differ from those of the films formed by the recipient strain (WT) B. subtilis 2335 (Km^s). However, the TM and the natural one differ in the dynamics of biofilm formation and the cellular composition of the films. Biofilms of the TM are formed earlier, develop at a higher rate, but decompose later than the films of the WT. When the medium is inoculated with vegetative cells, sporulation in the biofilms of both strains undergoes glucose repression; no such effect is observed when the medium is inoculated with spores. The TM does not form films when the medium is inoculated with spores and supplemented with glycerin and kanamycin.     

Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.     

A comparison between in vitro fertilization and microinjection of immobilized spermatozoa from bulls producing spermatozoa with defects.

The objectives of this study were to compare the fertilization rate of bovine in vitro matured oocytes by in vitro fertilization (IVF) and by microinjection of a single spermatozoon (MI) and to relate these rates with fertility reported for these bulls in artificial breeding. Bull A (Holstein) had a nonreturn rate of 75%. Semen from this bull is routinely used in our standard IVF procedure. Bull B (Ayrshire), used regularly in artificial breeding and related to bull D, had a nonreturn rate of 69.2%. Bull C (Brown Swiss), with a chromosomal translocation and trisomy, achieved a nonreturn rate of 42%. Bull D (Ayrshire) produced nonmotile spermatozoa (SPZ) and had an abnormality described as "tail stump defect." No pregnancies sired by bull D have been reported. Oocytes were either fertilized in vitro by capacitated SPZ or by microinjection of a single immobilized SPZ into the ooplasm. SPZ were treated with 0.1 microM A23187 and used for IVF. For microinjection SPZ were cocultured for 5 h with bovine oviduct epithelial cells (BOEC) and then immobilized by freezing and thawing twice without cryoprotectant. A single batch of killed SPZ (stored at -25 degrees C) was used for all microinjections. All oocytes were cultured in Medium 199 for 22 h at 39 degrees C and subsequently fixed, stained, and examined for evidence of fertilization (i.e., female and male pronucleus formation, SPZ decondensation). Fertilization rates following IVF with semen from bulls A, B, C, and D were 80%, 54%, 1%, and 2%, and following microinjection were 39%, 22%, 21%, and 34%, respectively.     

Inhibition of macropinocytosis blocks antigen presentation of type II collagen in vitro and in vivo in HLA-DR1 transgenic mice

Professional antigen-presenting cells, such as dendritic cells, macrophages and B cells have been implicated in the pathogenesis of rheumatoid arthritis, constituting a possible target for antigen-specific immunotherapy. We addressed the possibility of blocking antigen presentation of the type II collagen (CII)-derived immunodominant arthritogenic epitope CII_259–273 to specific CD4 T cells by inhibition of antigen uptake in HLA-DR1-transgenic mice in vitro and in vivo. Electron microscopy, confocal microscopy, subcellular fractionation and antigen presentation assays were used to establish the mechanisms of uptake, intracellular localization and antigen presentation of CII by dendritic cells and macrophages. We show that CII accumulated in membrane fractions of intermediate density corresponding to late endosomes. Treatment of dendritic cells and macrophages with cytochalasin D or amiloride prevented the intracellular appearance of CII and blocked antigen presentation of CII_259–273 to HLA-DR1-restricted T cell hybridomas. The data suggest that CII was taken up by dendritic cells and macrophages predominantly via macropinocytosis. Administration of amiloride in vivo prevented activation of CII-specific polyclonal T cells in the draining popliteal lymph nodes. This study suggests that selective targeting of CII internalization in professional antigen-presenting cells prevents activation of autoimmune T cells, constituting a novel therapeutic strategy for the immunotherapy of rheumatoid arthritis.     

Differences in protein phosphorylation in vivo and in vitro between wild type and dunce mutant strains of Drosophila melanogaster

The protein phosphorylation patterns of wild type and dunce mutant strains of Drosophila melanogaster, as detected by sodium dodecylsulfate-gel electrophoresis and autoradiography, have been compared. After labelling in vivo with 32Pi or in vitro in homogenates with [gamma-32P]ATP, radioactive bands at and above apparent polypeptide mol. wt approximately 110,000 were more pronounced in dunce fly heads than in wild type heads. When labelling in vitro, in dunceM11 there appeared a radioactive band at apparent mol. wt approximately equal to 53,000 that was faintly visible in the wild strain. The same band could be intensified in both strains by adding cyclic AMP to the homogenate or by performing homogenization in the presence of theophylline. The data suggest that the mol. wt approximately equal to 53,000 protein is a substrate for cyclic AMP-dependent protein kinase.     

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best‐characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild‐type poplar hybrid Populus tremula × P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding γ‐glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild‐type plants; soil contamination significantly decreased biomass accumulation in both wild‐type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two‐dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild‐type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal‐contaminated field sites. Further experiments are required to test these assumptions.     

Fertility of cooled and frozen rabbit sperm measured by competitive fertilization

The fertility of rabbit sperm that had been cooled to 5 degrees C or frozen and thawed was determined by competitive fertilization. Treatments were identified by labeling sperm with fluorescein isothiocyanate (FITC) or tetramethylrhodamine B isothiocyanate (TRITC). Sperm from different treatments were mixed and used in a competitive insemination experiment. Does were inseminated 5, 10 or 15 h prior to ovulation. Time of ovulation was controlled by injections of luteinizing hormone. The functional sperm transport, as determined by the number of sperm transported to the site of fertilization and capable of fertilizing oocytes, was estimated by counting the total number of differently stained sperm that surrounded or fertilized each oocyte. The fertility of sperm cooled to 5 degrees C was not affected (p less than 0.05) as compared to fertility of uncooled sperm. Functional sperm transport at all times of insemination and fertilization ratio at insemination 10 or 15 h before ovulation were reduced (p less than 0.05) for frozen-thawed vs. cooled sperm. No difference in fertilization ratio (p greater than 0.05) occurred, however, when does were inseminated 5 h before ovulation. While sperm survival and capacitation time appeared to play roles in fertility of frozen-thawed sperm, the most important factor was reduced functional sperm transport. However, fertility of frozen-thawed sperm was improved when the time from insemination to ovulation was reduced.     

Cytogenetic consequences of hybridization between wild and laboratory mice

Wild mice from the aboriginal population were crossed with the laboratory C57BL/6 mice. F1 hybrids were backcrossed with both parents. Significant increase of recombination frequency in the AY-Ra region of the 2nd chromosome (from 22.0 to 33.1 cM), of the frequency of occurrence of X-Y univalents (up to 54.6%) and of autosomal univalents (up to 9.1%) was found in the F1. In backcrosses all these induces decreased gradually.     

Axenic mycorrhization of wild type and transgenic hybrid aspen expressing T-DNA indoleacetic acid-biosynthetic genes

 We describe and document the in vitro synthesis of ectomycorrhiza between roots of wild type and transgenic aspen (Populus tremula × P. tremuloides), expressing Agrobacterium tumefaciens T-DNA indoleacetic acid (IAA)-biosynthetic genes, and Amanita muscaria. Plantlets were raised from tissue culture. The root system of approximately 4-week-old plantlets was transferred to Petri dishes and incubated together with fungal mycelia under sterile conditions. Ectomycorrhiza showing both a well developed hyphal mantle and Hartig net were established within 3 to 4 weeks. Formation and morphology of ectomycorrhiza were not affected by the transformation of aspen, expressing the IAA biosynthetic genes in roots. As both hybrid aspen and fungal cells can be genetically engineered, this system offers a new approach to the study of mycorrhizal symbioses. Received: 19 January 1996 / Accepted: 23 January 1996     

Studying and managing the risk of cross-fertilization between transgenic crops and wild relatives

Drawing on field studies of pollen dispersal, we identify features of the hybridization process that need quantification. Our emphasis is on standardized measures, as opposed to the idiosyncratic and often anecdotal methods with which gene flow or out-crossing data are currently reported. In addition to proposing specific maximum likelihood approaches, we summarize some results to date from small-scale field trials that bear on the risks anticipated for large-scale commercialization. We conclude that absolute containment of recombinant pollen or genes is unlikely if physical isolation is the only containment strategy. Because we conclude that the escape of transgenic pollen is inevitable, we argue that the focus of risk analysis should be shifted towards the 'invasiveness' of transgenic plants and 'mitigation' of their impact on natural, as well as agricultural systems.     

Cross fertilization in vivo and in vitro between three species of vesper mice, Calomys (Rodentia, Cricetidae)

Cross fertilization was tested between oocytes of Calomys callidus and spermatozoa from C. callidus, C. musculinus and C. laucha by both in vivo and in vitro insemination. After in vivo and in vitro insemination, respectively, percentages of oocytes fertilized were 68.8 and 46.6 (C. callidus X C. callidus), 20.3 and 12.7 (C. callidus X C. musculinus), 26.7 and 4.3 (C. callidus X C. laucha). Thus, the percentages obtained after in vitro insemination were always lower than those obtained with in vivo insemination. It was found that 23.9% and 44.4% of two-cell hybrid embryos were present in oviducts 30 hr after in vivo insemination of C. callidus females with C. musculinus or C. laucha spermatozoa, respectively. At a later stage (56 hr postinsemination), development did not progress further, and abnormal embryos were found both at 30 and 56 hr postinsemination, suggesting some kind of cleavage arrest or degeneration of the embryos. We suggest that fertilization is not strictly species-specific, at least among the species we studied, but that there are some factors that reduce the efficiency of interspecific fertilization.