Genetically stable cell lines of cucumber for the large-scale production of diploid somatic embryos

For the initiation of embryogenic cucumber ( Cucumis sativus L.) cell lines, from excised radicles, directly in liquid medium, the culture regime, explant density and type and concentration of hormones were adjusted so that pro-embryogenic masses (PEMs) were formed within about 8 weeks. The established cucumber cell lines were maintained tor several years without loss of embryogenic and genetic stability. The ploidy level of somatic embryos from different cucumber eell lines was either diploid or tetraploid and depended on the ploidy level of Ihe cell line. Cucumber cell lines that produced only diploid embryos were obtained by selecting completely diploid explant material and growing it in the dark during the initiation phase. Mixoploid explains could lead to tetraploid or mixoploid ceil lines. Isolation and additional selection and subculturing of single PEMs resulted in either completely diploid or tetraploid cell lines, indicating that all cells of individual PEMs are either diploid or tetraploid. The ernbryogenic cucumber cell Imes, differing only in ploidy level, were indistinguishable in growth rate and embryogetiic potential and were genetically stable over several years.     

Colonisation patterns of root tissues byPhytophthora nicotianae var.parasitica related to reduced disease in mycorrhizal tomato

Tomato plants pre-colonised by the arbuscular mycorrhizal fungusGlomus mosseae showed decreased root damage by the pathogenPhytophthora nicotianae var.parasitica. In analyses of the cellular bases of their bioprotective effect, a prerequisite for cytological investigations of tissue interactions betweenG. mosseae andP. nicotianae v.parasitica was to discriminate between the hyphae of the two fungi within root tissues. We report the use of antibodies as useful tools, in the absence of an appropriate stain for distinguishing hyphae ofP. nicotianae v.parasitica from those ofG. mosseae inside roots, and present observations on the colonisation patterns by the pathogenic fungus alone or during interactions in mycorrhizal roots. Infection intensity of the pathogen, estimated using an immunoenzyme labelling technique on whole root fragments, was lower in mycorrhizal roots. Immunogold labelling ofP. nicotianae v.parasitica on cross-sections of infected tomato roots showed that inter or intracellular hyphae developed mainly in the cortex, and their presence induced necrosis of host cells, the wall and contents of which showed a strong autofluorescence in reaction to the pathogen. In dual fungal infections of tomato root systems, hyphae of the symbiont and the pathogen were in most cases in different root regions, but they could also be observed in the same root tissues. The number ofP. nicotianae v.parasitica hyphae growing in the root cortex was greatly reduced in mycorrhizal root systems, and in mycorrhizal tissues infected by the pathogen, arbuscule-containing cells surrounded by intercellularP. nicotianae v.parasitica hyphae did not necrose and only a weak autofluorescence was associated with the host cells. Results are discussed in relation to possible processes involved in the phenomenon of bioprotection in arbuscular mycorrhizal plants.     

克鲁斯假丝酵母及其近似种的脉冲电泳核型分析

用钳位均匀电场脉冲电泳(CHEF)系统分析了克鲁斯假丝酵母(Candida krusei),郎比可假丝酵母(C. lambica)和粗状假丝酵母(C. valiad)的模式菌株的电泳核型,发现这三种表型相似的假丝酵母却具有互不相同的染色体DNA分子带型,为其分类学研究提供了可靠的鉴别依据。在常规分类学研究的基础上,测定了AS 2.75(原定种名为(C. incospicua),AS2.1182(原定种名为 C. lambica)和AS 2.1772(未定种)等三株假丝酵母的G+C含量和脉冲电泳核型。通过对已报道的C. inconspicu的G+C含量及上述三种假丝酵母模式菌株的脉冲电泳核型的比较分析证明,AS 2.75和AS 2.1772为粗状假丝酵母(C. valida),AS 2.1182为克鲁斯假丝酵母(C. krusei)。     

有机硅橡胶固定化细胞进行的生物转化

有机硅橡胶固定化细胞进行的生物转化潘冰峰,戴学倩,冯青,李祖义（中国科学院上海有机化学研究所,２０００３２）关键词硅橡胶;白地霉;固定化细胞;生物转化近年来,有机化学领域的一个重要进展是用酶或微生物进行生物转化反应。其中研究和应用较多的是碳基还原,特...     

钙调素对细胞周期的调节

RC3细胞是一种用真核表达载体1~(CaM)转染NIH 3T3细胞建成的可调钙凋素(Calmodulin,CaM)高表达细胞模型。通过分子杂交及蛋白免疫印迹方法证实在地塞米松(Dexamethasome,DXM)作用下,RC3细胞可高表达CaM。CaM的过表达使G_1期细胞减少,S期细胞增加;CaM拮抗剂三氟拉嗪(trifluoperazine,TFP)则使G_1期细胞增加,S期细胞减少。高表达CaM使细胞分裂指数提高,G_2期细胞减少,有丝分裂前期细胞增加,M中期细胞比例下降。而TFP处理则使分裂指数下降,G_2期细胞增加,M前期细胞减少,M中期细胞增加。实验结果表明CaM在G_1/S、G_2/M和M中期/M后期3个位点上对细胞周期进行调控;通过加速G_1至S期,G_2至M期和M中期至M后期的进程,使细胞倍增时间缩短,促进细胞增殖。本工作表明,RC3细胞作为CaM表达可调细胞模型,是研究细胞周期调控的有力工具。     

Phylogeny of Drosophila and related genera inferred from the nucleotide sequence of the Cu,Zn Sod gene

The phylogeny and taxonomy of the drosophilids have been the subject of extensive investigations. Recently, Grimaldi (1990) has challenged some common conceptions, and several sets of molecular data have provided information not always compatible with other taxonomic knowledge or consistent with each other. We present the coding nucleotide sequence of the Cu,Zn superoxide dismutase gene (Sod) for 15 species, which include the medfly Ceratitis capitata (family Tephritidae), the genera Chymomyza and Zaprionus, and representatives of the subgenera Dorsilopha, Drosophila, Hirtodrosophila, Scaptodrosophila, and Sophophora. Phylogenetic analysis of the Sod sequences indicates that Scaptodrosophila and Chymomyza branched off the main lineage before the major Drosophila radiations. The presence of a second intron in Chymomyza and Scaptodrosophila (as well as in the medfly) confirms the early divergence of these two taxa. This second intron became deleted from the main lineage before the major Drosophila radiations. According to the Sod sequences, Sophophora (including the melanogaster, obscura, saltans, and willistoni species groups) is older than the subgenus Drosophila; a deep branch splits the willistoni and saltans groups from the melanogaster and obscura groups. The genus Zaprionus and the subgenera Dorsilopha and Hirtodrosophila appear as branches of a prolific “bush” that also embraces the numerous species of the subgenus Drosophila. The Sod results corroborate in many, but not all, respects Throckmorton's (King, R.C. (ed) Handbook of Genetics. Plenum Press, New York, pp. 421–469, 1975) phylogeny; are inconsistent in some important ways with Grimaldi's (Bull. Am. Museum Nat. Hist. 197:1–139, 1990) cladistic analysis; and also are inconsistent with some inferences based on mitochondrial DNA data. The Sod results manifest how, in addition to the information derived from nucleotide sequences, structural features (i.e., the deletion of an intron) can help resolve phylogenetic issues. Correspondence requests to: F. J. Ayala     

Compositional heterogeneity of the Escherichia coli genome: A role for VSP repair?

E. coli genes that contain a high frequency of the tetranucleotide CTAG are also rich in the tetramers CTTG, CCTA, CCAA, TTGG, TAGG, and CAAG (group-I tetramers). Conversely, E. coli genes lacking CTAG are rich in the tetranucleotides CCTG, CCAG, CTGG, and CAGG (group-II tetramers). These two gene samples differ also in codon usage, amino acid composition, frequency of Dcm sites, and contrast vocabularies. Group-I tetramers have in common that they are depleted by very-short-patch repair (VSP), while group-II tetramers are favored by VSP activity. The VSP system repairs G:T mismatches to G:C, thereby increasing the overall G+C content of the genome; for this reason the CTAG-rich sample has a lower G+C content than the CTAG-poor sample. This compositional heterogeneity can be tentatively explained by a low level of VSP activity on the CTAG-rich sample. A negative correlation is found between the frequency of group-I tetramers and the level of gene expression, as measured by the Codon Adaptation Index (CAI). A possible link between the rate of VSP activity and the level of gene expression is considered.Correspondence to: A. Marine     

Modulation of Adenylylcyclase by Protein Kinase C in Human Neurotumor SK-N-MC Cells: Evidence that the α Isozyme Mediates Both Potentiation and Desensitization

Abstract: Exposure of human SK-N-MC neurotumor cells to 4β-phorbol 12-myristate 13-acetate (PMA) increased isoproterenol stimulation of cyclic AMP levels by severalfold. This potentiation was blocked by inhibitors of protein kinase C (PKC) and did not occur in cells in which PKC had been down-regulated. PMA treatment also enhanced the stimulation by dopamine, cholera toxin, and forskolin. Thus, the effect of PMA on the adenylylcyclase system was postreceptor and involved either the guanine nucleotide binding regulatory (G) proteins or the cyclase itself. As PMA treatment did not impair the inhibition of isoproterenol stimulation by neuropeptide Y, an involvement of the inhibitory G protein G_i was unlikely. Cholate extracts of membranes from control and PMA-treated cells were equally effective in the reconstitution of adenylylcyclase activity in S49 cyc^? membranes, which lack the stimulatory G protein subunit G_sα; thus, G_s did not appear to be the target of PMA action. Membranes from PMA-treated cells exhibited increased adenylylcyclase activity to all stimulators including Mn²⁺ and Mn²⁺ plus forskolin. In addition, activity was increased when control membranes were incubated with ATP and purified PKC from rat brain. This is consistent with a direct effect of PKC on the adenylylcyclase catalyst in SK-N-MC cells. PMA treatment also resulted in a shift to less sensitivity in the K_act for isoproterenol but not for dopamine or CGP-12177 (a β₃-adrenergic agonist) stimulation. Thus, the β₁ but not the D₁ or β₃ receptors were being desensitized by PKC activation. Analysis of SK-N-MC cells by western blotting with antibodies against different PKC isozymes revealed that both the α and ζ isozymes were present in these cells. Whereas PKC-α was activated and translocated from cytosol to membrane by phorbol esters, the ζ isozyme was not. Thus, PKC-α, which has been implicated in desensitization in other cell lines, also appears to potentiate adenylylcyclase activity.     

Differential Requirements of Sodium for Coupling of Cannabinoid Receptors to Adenylyl Cyclase in Rat Brain Membranes

Abstract: Sodium is generally required for optimal inhibition of adenylyl cyclase by G_l/o-coupled receptors. Canna-binoids bind to specific receptors that act like other members of the G_l/o-coupled receptor superfamily to inhibit adenylyl cyclase. However, assay of cannabinoid inhibition of adenylyl cyclase in rat cerebellar membranes revealed that concentrations of NaCI ranging from 0 to 150 mM had no effect on agonist inhibition. This lack of effect of sodium was not unique to cannabinoid receptors, because the same results were observed using baclofen as an agonist for GABA_B receptors in cerebellar membranes. The lack of sodium dependence was region-specific, because assay of cannabinoid and opioid inhibition of adenylyl cyclase in striatum revealed an expected sodium dependence, with 50 mM NaCI providing maximal inhibition levels by both sets of agonists. This difference in sodium requirements between these two regions was maintained at the G protein level, because agonist-stimulated low K_m GTPase activity was maximal at 50 mM NaCI in striatal membranes, but was maximal in the absence of NaCI in cerebellar membranes. Assay of [³H]WIN 55212–2 binding in cerebellar membranes revealed that the binding of this labeled agonist was sensitive to sodium and guanine nucleotides like other G_l/o-coupled receptors, because both NaCI and the nonhydrolyzable GTP analogue Gpp(NH)p significantly inhibited binding. These results suggest that differences in receptor-G protein coupling exist for cannabinoid receptors between these two brain regions.     

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.