Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion

Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (Kan^R) and -glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several Kan^R calli. A high frequency of plants regenerated from Kan^R calli expressed both Kan^R and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.     

Production and identification of somatic hybrids between Solanum tuberosum and S. papita by using the rolC gene as a morphological selectable marker

A successful hybridization of a diploid clone of Solanum tuberosum with a rolC-transgenic, diploid S. papita clone is reported. By using leaf expiants of this S. papita clone, which after transformation expressed kanamycin resistance, intact protoplasts were obtained, but these protoplasts did not develop to microcalli or regenerate to mature plants. However, protoplasts of the S. tuberosum clone showed a high capacity to regenerate plants from isolated protoplasts. On a medium containing Kanamycin only calli regenerated to plants, which revealed a rolC phenotype (reduced apical dominance with a large number of adventitious shoots and a pale green color of leaves) and later on turned out to be true hybrids. Self fusions of S. papita never developed to microcalli and those of S. tuberosum ceased to develop on the kanamycin-containing medium. Identification of somatic hybrids was done by RFLP and RAPD analysis. In the greenhouse, out of four selected hybrids only FK3.1 was successfully crossed with two standard S. tuberosum varieties (Datura, Desirée). Out of all the seeds germinated, only rolC-negative F¹ seedlings were further characterized. Within the seedling population obvious differences were evident in respect of the S. papita and S. tuberosum characteristics.     

Development of human monoclonal antibodies: A review

This article describes the current status in the development of human monoclonal antibodies. Over the last ten years a lot of information about the human immune system has emerged. Combining these with the many new (bio-)technologies it is plausible that the long awaited breakthrough of this technology is close. This paper focuses on the classical cell-biological methods of achieving stable, antibody-producing human cell lines via cell fusion methods or virus derived transformations of human B-lymphocytes, as well as genetic engineering methods e.g. DNA libraries or phage display technology. The available in vitro immunization methods are critically reviewed and their impact on this topic is discussed. Therapeutic applications for cancer treatment or passive immunization against infectious diseases with antibodies derived by both ways are also reviewed.     

CHROMOSOMAL VERSUS MITOCHONDRIAL DNA EVOLUTION: TRACKING THE EVOLUTIONARY HISTORY OF THE SOUTHWESTERN EUROPEAN POPULATIONS OF THE SOREX ARANEUS GROUP (MAMMALIA,INSECTIVORA)

The shrews of the Sorex araneus group have undergone a spectacular chromosome evolution. The karyotype of Sorex granarius is generally considered ancestral to those of Sorex coronatus and S. araneus. However, a sequence of 777 base pairs of the cytochrome b gene of the mitochondrial DNA (mtDNA) produces a quite different picture: S. granarius is closely related to the populations of S. araneus from the Pyrenees and from the northwestern Alps, whereas S. coronatus and S. araneus from Italy and the southern Alps represent two well-separated lineages. It is suggested that mtDNA and chromosomal evolution are in this case largely independant processes. Whereas mtDNA haplotypes are closely linked to the geographical history of the populations, chromosomal mutations were probably transmitted from one population to another. Available data suggest that the impressive chromosome polymorphism of this group is quite a recent phenomenon.     

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca²⁺] _o (extracellular Ca²⁺ concentration) is between about 10^–4 to 10^–3 m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca²⁺ _o or increasing [Ca²⁺] _o to 10^–2 m inhibit exocytotic membrane fusion. Veratridine does not release Ca²⁺ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na⁺ _o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca²⁺] _o ) in the somatic, i.e., nonciliary cell membrane and that a Ca²⁺ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.We thank Dr. C. Kung (Madison, WI) for providing the pawn mutant, Drs. G. Lehle and R. Waldschütz-Schüppel (Konstanz, Germany) for their help with light scattering experiments, and Ms. E. Dassler and D. Bliestle for continuous help during the extensive photographic documentation. This work has been supported by Deutsche Forschungsgemeinschaft, Schwerpunkt Neue mikroskopische Techniken für Biologie und Medizin (grant P178/11) and SFB156/B4.     

Cybridization in Nicotiana tabacum L. using double inactivation of parental protoplasts and post-fusion selection based on nuclear-encoded and chloroplast-encoded marker genes

An effective selection system preceded by double inactivation of parental protoplasts was used to transfer Nicotiana suaveolens Leh. cytoplasmic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cultivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens plants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inactivated with iodoacetamide or rhodamine 6G, while those of the cytoplasm donor were inactivated by X-irradiation. The resultant microcalli were cultured on a shoot regeneration medium containing both kanamycin and spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants, out of a total of 44 regenerated plants transferred to the glasshouse, were obtained. Intraspecific somatic transfers of the CMS trait between N. tabacum cultivars with distinctlydifferent morphologies using single inactivation and nonselective shoot regeneration medium were demonstrated. The implications of the results for practical tobacco breeding as a means of circumventing lengthy backcrossing procedures are discussed.     

Approach to the Involvement of Influenza B Neuraminidase in the Cleavage of HA by Host Cell Protease Using Low pH-Induced Cell Fusion Reaction

ts7, a temperature-sensitive mutant defective in neuraminidase (NA) of influenza B/Kanagawa/73, lacks NA enzymatic activity at the nonpermissive temperature (37.5 C). When MDCK cells were infected with the mutant at the permissive temperature (32 C) and exposed to pH 5.2 medium, extensive cell fusion occurred. In contrast, at the nonpermissive temperature cells did not show cell fusion at all unless they were pretreated with trypsin, suggesting that at 37.5 C the hemagglutinin (HA) of ts7 is expressed at the cell surface in an uncleaved form. It was also found that the replacement of RNA segment 6 of ts7 with that of wild-type B/Lee resulted in the emergence of low pH-induced fusion activity as well as NA enzymatic activity at the incubation temperature of 37.5 C and that the addition of bacterial NA to the cultures infected with ts7 at 37.5 C early in infection brought about low pH-induced cell fusion. We suggest that the removal of neuraminic acid from the carbohydrate moiety of HA by NA is essential for the cleavage of HA by cellular protease.     

新型双分子细胞因子融合蛋白研究进展

细胞因子通过相互协调、相互制约在体内发挥着重要的免疫调节作用,利用细胞因子的这一特点,近年来国内外设计并构建了新型的第二代细胞因子,即利用基因工程技术和蛋白质工程技术将两种细胞因子合二为一,使之成为具有多功能的嵌合蛋白新品种,为细胞因子的理论研究及临床应用提供了新的手段与方法.     

人重组IL6／IL2融合蛋白的变性、复性及纯化

经超声破碎,分离已表达CH925包涵体,较系统地研究变性剂浓度、融合蛋白浓度对蛋白折叠的影响.在还原型及氧化型谷胱甘肽复性条件下,成功地将融合蛋白CH925折叠成具有IL6及IL2双活性蛋白,IL6的比活为2.3×10⁸U/mg, IL2比活为2.2×10⁶U/mg.经阴离子交换、凝胶过滤层析,获得一定纯度的CH925,配合反相HPLC.洗脱收集蛋白峰,CH925纯度为98%.     

Special features of phosphatidylcholine vesicles as seen in cryo-transmission electron microscopy

Vesicles of egg yolk phosphatidylcholine (EYPC) were studied by cryo-transmission electron microscopy. The electron micrographs indicate that, despite the rapidity of cooling, membrane undulations are flattened and some vesicles change their shapes before the samples freeze. These artefacts are attributed to the action of the lateral tension that results from the membrane area contraction associated with the temperature drop. Other micrographs represent grainy membranes and angular vesicles. We regard them as the first direct evidence for the superstructure and optically invisible roughness which were recently postulated for these membranes.