生物素肼化学标记和DIG标记cDNA探针检测柑桔裂皮病类病毒

分别用生物素肼化学标记和ＤＩＧ标记我国柑桔裂皮病类病毒（ＣＥＶｄ）全长克隆ｃＤＮＡ,用以上探讨对不同来源的的核酸样品进行斑点杂交。两种探针可检出病柑桔总核酸的最低含量久为４００ｎｇ／斑点和８０ｎｇ／斑点;生物素肼标记ＣＥＶｄ－ＤＮＡ探讨针,可检出ＣＥＶｄ－ｃＤＮＡ的最低含量约为１０ｐｇ／斑点,研制的ＣＥＶｄ检测试剂盒能检测出发病和隐性带毒苗木中的ＣＥＶｄ,灵敏、特异、简便、快速。试剂盒已使用于检测     

酶标DNA探针与Y染色体DNA的探测

 用辣根过氧化物酶标记DNA的技术,制备了酶标基因探针。研究了酶标过程和产物的电泳行为;用斑点杂交和southern印迹杂交探测了单链、双链DNA,灵敏度可达pg水平,以此酶标的Y染色体特异的DNA片段作探针,进行了DNA杂交的性别分析,证明该探针能清楚地区别两性基因组DNA,这对基因的研究和诊断有一定实用价值。     

爪哇三七组织培养类病毒的初步研究

用含裂皮病类病毒(Citrus exoeortis viroid,简称CEV)的古巴花叶橙、冰糖橙的叶汁和核酸粗提物感染爪哇三七(Gynura aurantiaca),取其腋芽和嫩叶作外植体,经消毒处理,接种在稍加改良的B5和MS半固体培养基上,置于24℃～27℃遮光培养,经2～3周诱导形成愈伤组织,质地有的松散易碎似白木耳状,有的坚硬灰黑似多瘤状,经双向凝胶电泳-银染法和点杂交法检测,证实用CEV感染的嫩叶和腋芽作外植体,不仅能诱导形成愈伤组织,而且CEV还可能在愈伤组织中复制,结果表明,已初步建立了爪哇三七的组织培养和研究类病毒的离体培养系统。     

~(60)Co-γ辐照对中国仓鼠卵巢成纤维细胞DNA和组蛋白合成的影响

本文介绍了~(60)Co-γ辐照对同步的和非同步的CHO细胞的DNA合成和组蛋白合成关系的影响的研究,用~3H-胸腺嘧啶核苷和~(14)C-丙氨酸双标记,未经辐照的和经4Gy～60Gy ~(60)Co-γ辐照的CHO细胞,通过~3H和~(14)C的参入来估价DNA和组蛋白的合成,并用聚丙烯酰胺凝胶电泳鉴定辐照前后组蛋白各组分的变化情况,实验表明: 1)、在4～60Gy剂量范内,无论是同步的还是非同步的CHO细胞其DNA合成和组蛋白合成都受到不同程度的抑制。2)、在辐照后1—3小时,DNA合成和组蛋白合成都受到不同程度的抑制,但辐照后4小时,DNA合成被进一步抑制而组蛋白的合成却逐渐恢复正常,到辐照后48小时组蛋白的合成几乎接近对照水平。3)、16Gy ~(60)Co-γ辐照后8小时,非同步的CHO细胞的DNA合成被抑制的情况比G_1期CHO细胞更为严重。4)、16Gy ~(60)Co-γ辐照S期细胞,在辐照后1—24小时中DNA合成被明显抑制的同时,组蛋白的合成也受到相应的抑制。5)、从未经辐照的和经6、16和60Gy~(60)Co-γ辐照的CHO细胞分别提取全组蛋白,进行聚丙烯酰胺凝胶电泳,从电泳图谱的变化清楚地看到组蛋白H_1和H_3受辐照影响大于组蛋白H_4和H_(2B)+H_(2A),因此我们推测DNA合成和组蛋白H_1和H_3的关系较之组蛋白H_4和H_(2A)+H_(2B)更为密切。     

DNA PROBES AND A RECEPTOR-BINDING ASSAY FOR DETECTION OF PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) SPECIES AND DOMOIC ACID ACTIVITY IN CULTURED AND NATURAL SAMPLES

Large-subunit ribosomal RNA-targeted probes for Pseudo-nitzschia australis Frenguelli, P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, and P. pungens (Grunow) Hasle were applied to cultured and natural samples using whole-cell and sandwich hybridization. Testing of the latter method is emphasized here, and technique refinements that took place during 1996–1997 are documented. Application of the sandwich hybridization test showed that the signal intensity obtained for a given number of target cells remained constant as batch cultures of these organisms progressed from active through stationary growth phases. This suggests that cellular rRNA content for each target species remained relatively stable despite changes in growth state. Application of whole-cell and sandwich hybridization assays to natural samples showed that both methods could be used to detect wild P. australis, P. pseudodelicatissima, and to a lesser degree P. multiseries, but detection of P. pungens was prone to error. A receptor-binding assay for domoic acid (DA) enabled detection of this toxin activity associated with a particulate fraction of the plankton and provided a context in which to view results of the rRNA probe tests. In one case, the probe for P. australis cross-reacted with P. cf. delicatissima. The sample that contained the latter species also contained a low amount of DA activity. Under certain field conditions, results of whole-cell and sandwich hybridization tests disagreed. Detailed analysis of selected field samples illustrates how such situations arose. Collectively, the rRNA probe and toxin analyses suggest that manifestation of DA in the environment is possible in the absence of readily recognizable intact cells.     

地高辛标记反意RNA探针检测脑组织切片生长抑素mRNA

本实验采用含大鼠生长抑素基因的ｐＳＰ６５ｃＤＮＡ质位通过转化至噬菌体内大量扩增,经提取,纯化后,用限制性内切酶进行酶切使质枝线性化,并将其作为模板,用地高辛（ＤｉｇｏｘｉｇｅｎｉｎＤｉｇ）作为标记物,体外转录合成生长抑素反意ＲＮＡ（ｃＲＮＡ）探针。实验动物选用ｗｉｓｔａｒ新生大鼠。冰冻切片,端、间脑切片经杂交前用Ｄｉｇ－ＵＴＰ标记的ｃＲＮＡ探针杂交,杂交后用抗Ｄｉｇ－碱性磷酸酶复合物进行酶联免疫反应。Ｘ－磷酸盐－ＮＢＴ显色。结果显示新生大鼠脑内生长抑素ｍＲＮＡ神经元着紫蓝色。杂交反应物集中于核周的胞浆及短小的突起内。胞核不着色。胞体轮廓清晰,周围背底浅淡。结果表明Ｄｉｇ标记ｃＲＮＡ探针不仅具备非同位素标记探针的优点而且能快速和准确检测组织细胞内ｍＲＮＡ的表达。     

~(90)锶对鲫鱼胚胎发育的影响及鲫仔鱼对~(90)锶吸收积累的研究

在胚胎发育过程中,用不同比放射性的~(90)锶试液处理受精卵,各处理组都有畸形和死亡现象。畸形的高峰一般出现在囊胚期和晶体形成期,畸形的类型一般为脊椎弯曲、体短、胸腔和卵黄囊扩大,头和眼畸形。随后即出现死亡高峰。例如:游动期的死亡率分别是:21%(对照);36%(5×10~(-11)居里/升);40%(5×1-~(-10)居里/升);45%(5×10~(-9)居里/升)。鲫仔鱼对~(90)锶吸收积累的结果表明:鲫仔鱼体中比放射性大小与试液的比放射性大小成正比,并随试验吋间的延长,生物量的增加而增加,鲫仔鱼的积累系数则不依试液的比放射性大小和生物量的多少为转移。     

紫外线^60Co照射对蛇毒毒性的影响

选择体重20g 左右小白鼠分别使用眼镜蛇毒、蝮蛇毒以及经过紫外线、~(60)Co 照射灭菌后的这两种蛇毒进行半数致死量的测定,结果经紫外线照射后的眼镜蛇毒和白眉蝮蛇毒的毒性降低了6.98%和6.41%.经~(60)Co 照射的眼镜蛇毒和蝮蛇毒的毒性降低了21%和33%.     

应用聚合酶链式反应扩增和光敏生物素标记的cDNA探针检测马铃薯纺锤块茎类病毒

以含马铃薯纺锤块茎类病毒(potato spindle tuber viroid,PSTV)RNA的总核酸为模板,加入人工合成的互补DNA引物,用反转录酶合成PSTV cDNA;在聚合酶链式反应系统中,用两个PSTV特异性引物进行cDNA扩增,用以制备光敏生物素标记的PSTV cDNA探针。用此探针进行斑点杂交检测含PSTV的马铃薯核酸提取液和汁液均出现阳性杂交信号,而健康马铃薯的核酸提取液和汁液的结果均为阴性。光敏生物素标记探针检测纯化PSTV的灵敏度可达5pg;检测感染PSTV的马铃薯块茎汁液的可测出最高稀释度为1:400。     

IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM CATENELLA AND A. TAMARENSE (DINOPHYCEAE) USING DNA PROBES AND WHOLE-CELL HYBRIDIZATION1

Fluorescent DNA probes (cCAT-F1 and cTAM-Fl) complementary to the 3′ end of ribosomal RNA (rRNA) internal transcribed spacer 1 sequences (ITS 1: positions 154–176) of toxic species of Alexandrium catenella (Whedon and Kofoid) Taylor and A. tamarense (Lebour) Taylor were applied to various cultures of the genus Alexandrium and several other phytoplankters using whole-cell fluorescent in situ hybridization. cCAT-F1 and cTAM-F1 reacted with targeted strains of A. catenella (catenella type) and A. tamarense (tamarense type), respectively, and did not react with isolates of A. affine (Inoue et Fukuyo) Balech, A. fraterculus (Balech) Balech, A. insuetum Balech, A. lusitanicum Balech, A. pseudogonyaulux (Biecheler)Horiguchi ex Yuki et Fukuyo comb. nov., nor isolates of Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra (Ehrenberg) Stein, Gymnodinium mikimotoi Miyake et Kominami ex Oda, Skeletonema costatum (Greville) Cleve, Heterosigma akashiwo (Hada) Hada, and Chattonella antiqua (Hada) Ono. DNase I and RNase A treatment showed that probes hybridized to ribosomal DNA, not rRNA. Probes were localized at the bottom of the U-shaped nucleus, a region that corresponds to the nucleolus. The probes are highly specific for particular strains of A. catenella and A. tamarense and are applicable for identifying these species collected from cultured and possibly natural populations.     

小麦根圈细菌铁载体的检测

本文报道微生物铁载体定性与定量测定的方法,以及小麦根圈细菌铁载体的室内检测结果。２２株小麦根圈耐寒细菌和２株固氮菌在定性检测平板上产生铁载体,另有３株固氮菌不产。定量测定结果表明：３６株水溶性色素产生菌合成铁载体的量在０．７１５－０．１０６（Ａ／Ａｒ）之间,其中属于荧光假单胞菌群的２５个菌株铁载体产量的测定值（Ａ／Ａｒ）在０．１８４－０．１０６之间,达极高量水平。     

DETECTION OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM FUNDYENSE (DINOPHYCEAE) WITH OLIGONUCLEOTIDE AND ANTIBODY PROBES: VARIABILITY IN LABELING INTENSITY WITH PHYSIOLOGICAL CONDITION

The toxic dinoflagellate Alexandrium fundyense Balech was grown under temperature- and nutrient-limited conditions, and changes in labeling intensity on intact cells were determined for two probe types: an oligonucleotide probe targeting rRNA and a monoclonal antibody (MAb) targeting a cell surface protein. In nutrient-replete batch culture, labeling with the rRNA probe was up to 400% brighter during exponential phase than during stationary phase, whereas MAb labeling did not change significantly with growth stage at the optimal growth temperature. In cultures grown at suboptimal, low temperatures, there was a significant difference between labeling intensity in stationary versus exponential phase for both probe types, with exponential cells labeling brighter with the rRNA probe and slightly weaker with the MAb. The decrease in rRNA probe labeling with increasing culture age was likely due to lower abundance of the target nucleic acid, as extracted RNA varied in a similar manner. With the MAb and the rRNA probes, slower growing cultures at low, nonoptimal temperature labeled 35% and 50% brighter than cells growing faster at warmer temperatures. Some differences in labeling intensity per cell disappeared when the data were normalized to surface area or volume, which indicated that the number of target antigens or rRNA molecules was relatively constant per unit area or volume, respectively. Slow growth accompanying phosphorus and nitrogen limitation resulted in up to a 400% decrease in labeling intensity with the rRNA probe compared to nutrient-replete levels, whereas the MAb labeling intensity increased by a maximum of 60%. With both probes, labeling was more intense under phosphorus limitation than under nitrogen limitation, and for all conditions tested, labeling intensity was from 600% to 3600% brighter with the MAb than with the rRNA probe. Thus, it is clear that significant levels of variability in labeling intensity can be expected with both probe types because of the influence of environmental conditions and growth stage on cellular biochemistry, cell size,rRNA levels, and the number or accessibility of cell surface proteins. Of the two probes tested, the rRNA probe was the most variable, suggesting that in automated, whole-cell assays, it can be used only in a semiquantitative manner. For manual counts, the human eye will likely accommodate the labeling differences. The MAb probe was less variable, and thus should be amenable to both manual and automated counts.     

THE TEMPORAL SCHEDULE OF DNA SYNTHESIS AND CELL DIFFERENTIATION

The temporal schedule of DNA synthesis in cells of developing and adult mice is analysed by means of Feulgen cytofluorometry combined with tritiated thymidine autoradiography. The results obtained with cells taken from liver, esophageal epithelium and mucosae of gastrointestinal tracts seemed to conform to the hypothesis that a cell at a particular state of cytodifferentiation possesses specifically inactivated sets of late replicating genes showing a specific pattern of the temporal schedule of DNA synthesis.