LARGE-SCALE ISOLATION AND FRACTIONATION OF ORGANS OF DROSOPHILA MELANOGASTER LARVAE

Methods for the mass isolation of diverse organs from small animals are described. They involve novel devices: a mechanical dissecting system, a centrifugal agitator for the separation of fibrillar from globular particles, and a settling chamber for the fractionation at unit gravity of particles with sedimentation velocities above the useful range for centrifugation. The application of these methods to the isolation of polytene and nonpolytene nuclei from Drosophila melanogaster larvae is described.     

CHARACTERIZATION OF AN α-BUNGAROTOXIN BINDING COMPONENT FROM DROSOPHILA MELANOGASTER

Abstract— We describe an α-bungarotoxin binding component from Dromphila melanoyaster that has the properties expected of an acetylcholine receptor. Toxin binding to a paniculate form of this component has been shown to be proportional to amount of extract, to be saturable and to be destroyed by heat. Localization studies using ¹²⁵I-α-bungarotoxin binding to frozen sections has shown toxin binding to be restricted to synaptic areas of the Drosophila CNS. We have also shown that this toxinbinding component can be treated with Triton X-100 without significantly altering its toxin-binding and pharmacological specificity. The ability of preincubation with cholinergic ligands to block labeled α-bungarotoxin binding to both particulate and detergent treated extracts has been studied. The nicotinic agents nicotine, d-tubocurarine, and acetylcholine are the most effective blocking agents. All of the muscarinic agents tested and the nicotinic agent decamethonium were less effective than acetylcholine in preventing α-bungarotoxin binding.     

几种濒危植物及其近缘类群总DNA的提取与鉴定

用低ｐＨ 介质,高盐沉淀蛋白质方法成功地从银杉（Ｃａｔｈａｙａ ａｒｇｙｒｏｐｈｙｌｌａ Ｃｈｕｎ ｅｔＫｕａｎｇ）、矮牡丹（Ｐａｅｏｎｉａ ｓｕｆｆｒｕｔｉｃｏｓａ ｖａｒ． ｓｐｏｎｔａｎｅａ）、南川升麻（Ｃｉｍ ｉｃｉｆｕｇａ ｎａｎｃｈｕａｎｅｎｓｉｓＨｓｉａｏ）、裂叶沙参（Ａｄｅｎｏｐｈｏｒａ ｌｏｂｏｐｈｙｌｌａ）的同属种泡沙参（Ａ． ｐｏｔａｎｉｎｉｉ）等植物中提取和部分纯化了细胞总ＤＮＡ,并对其产率、质量和纯度作了鉴定。此方法的关键是用了一个低ｐＨ提取介质,它能有效防止组织破碎及沉淀大量材料时的电离化作用及酚化合物的进一步氧化。所得ＤＮＡ 不需经氯化铯梯度离心或柱层析,直接可用于限制性片断长度多态性（ＲＦＬＰ）及随机扩增的ＤＮＡ多态性（ＲＡＰＤ）等分子水平的遗传标记。为检测濒危植物的遗传多样性提供了一套迅速、简便和可靠的技术方案     

PURIFICATION OF CHOLINE ACETYLTRANSFERASE FROM DROSOPHILA MELANOGASTER

Abstract— Purification of choline acetyltransferase (ChAc) from heads of Drosophila melanogaster , the richest known source of ChAc, has been accomplished. The stability of the enzyme was preserved by working with a concentration of protein above 0.1 mg/ml. The purification was carried out with ammonium sulfate fractionation and column chromatography on QAE-Sephadex, CoA-Sepharose, G-200 Sephadex, and PCMB-Sepharose. In a procedure using 100 g of Drosophila heads, the specific activity of the crude homogenate was 0.028 μmol/min/mg protein and that of the final product was 43 μmol/min/mg protein, representing a 1500 fold purification. A single protein band, containing all of the ChAc activity, was seen by polyacrylamide gel electrophoresis. A sharp pH optimum at 7.2 was observed. Apparent K_m's for acetyl CoA and choline were 90 μM and 47 μM , respectively. The molecular weight was determined to be 69,000. Isoelectric focusing of extracts of Drosophila heads showed only one peak of choline acetyltransferase activity with an apparent pi of 5.1.     

四棱豆根瘤菌的分离及特性

从四棱豆根瘤上分离出一株慢生型根瘤菌(Rhizolia Ps)。在显微镜下,该菌株为革兰氏阴性,有一根亚极生鞭毛,大小为0.59×1.49微米。其增代时间为15.52小时,在39℃和含1.5％NaCl培养基中均不能生长。回接时能使四棱豆幼苗根部结瘤,固氮酶活性为2.81微摩尔乙烯·克鲜瘤~(-1)·小时~(-1)。本文对该菌株的含碳化合物利用、B.T.B.反应、在肉膏蛋白胨上的生长情况、石蕊牛奶反应、淀粉和明胶水解等生理生化特征进行了试验。     

ISOLATION AND CHARACTERIZATION OF CHROMATIN FROM NEUROSPORA CRASSA

Different preparations of chromatin isolated from mycelia of Neurospora crassa were analyzed for DNA-associated RNA and proteins. The UV absorption spectra, the ultrastructure of chromatin, and the amino acid composition of the acid-extractable proteins were studied. The protein:DNA ratios range from 1.5 to 2.8; the RNA:DNA ratios range from 0.5 to 1.24. UV absorption shows a macimum at 259 mµ and a minimum at 238–239 mµ. The E280/E260 ranges from 0.59 to 0.70. Electron microscopy reveals a fibrous structure with individual fibers of 120–150 A average diameter. Attempts were made to study the protein by polyacrylamide gel electrophoresis and amino acid analysis. The results indicate that Neurospora chromatin does not contain basic proteins comparable to calf thymus histone. The ratios of basic to acidic amino acids range from 0.93 to 1.19. On electrophoresis, no bands are seen whose positions correspond to those of histones. Staining for basic proteins with fast green or eosin Y at pH 8.2 also shows a negative reaction, suggesting the absence of histones.     

THE GENETIC BASIS OF SEXUAL ISOLATION BETWEEN DROSOPHILA MELANOGASTER AND D. SIMULANS

The genetic analysis of sexual isolation between the closely-related species Drosophila melanogaster and Drosophila simulans involved two experiments with no-choice tests. The efficiency of sexual isolation was measured by the frequency of courtship initiation and interspecific mating. We first surveyed the variation in sexual isolation between D. melanogaster strains and D. simulans strains of different geographic origin. Then, to investigate variation in sexual isolation within strains, we made F₁ diallel sets of reciprocal crosses within strains of D. melanogaster and D. simulans. The F₁ diallel progeny of one sex were paired with the opposite sex of the other species. The first experiment showed significant differences in the frequency of interspecific mating between geographic strains. There were more matings between D. simulans females and D. melanogaster males than between D. melanogaster females and D. simulans males. The second experiment uncovered that the male genotypes in the D. melanogaster diallel significantly differed in interspecific mating frequency, but not in courtship initiation frequency. The female genotypes in the D. simulans diallel were not significantly different in courtship initiation and interspecific mating frequency. Genetic analysis reveals that in D. melanogaster males sexual isolation was not affected by either maternal cytoplasmic effects, sex-linked effects, or epistatic interaction. The main genetic components were directional dominance and overdominance. The F₁ males achieved more matings with D. simulans females than the inbred males. The genetic architecture of sexual isolation in D. melanogaster males argues for a history of weak or no selection for lower interspecific mating propensity. The behavioral causes of variation in sexual isolation between the two species are discussed.     

草鱼和鲤鱼线粒体 DNA 的分离纯化及其 COI 基因的分子克隆

本文报道配合使用差速离心和DNaseI核酸酶处理等步骤,从草鱼和鲤鱼新鲜肝组织分离线粒体DNA(mtDNA)的实验方法。这种方法经济简便,纯化的mtDNA产率多,纯度高,经限制性内切酶消化后进行琼脂糖凝胶电泳分离,可以得到清晰的DNA片段谱带,并可直接用于构建酶切图谱和线粒体基因的分子克隆。用这样的mtDNA,我们已克隆了草鱼和鲤鱼的细胞色素氧化酶亚基I基因(COI基因)。     

ISOLATION AND CHARACTERIZATION OF LUMINAL MEMBRANES FROM URINARY BLADDER

A method is reported for the isolation of a highly purified fraction of urinary bladder membranes containing hexagonal plaques. The method uses zonal centrifugation as the final step of fractionation. The purified fraction was characterized by its electron microscopic morphology, by its enzymatic profile, by quantitative and qualitative analysis of lipids and by the protein pattern obtained by electrophoresis in polyacrylamide sodium dodecyl sulfate gels. The fraction contains 65% lipids and 35% proteins. The major protein component has a molecular weight of 27,000 daltons. Phospholipids are more than the 54% of the total lipid weight. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol are the major phospholipids with 50%, 30%, and 7% of the total lipid phosphorus, respectively. The glycolipid fraction is 10% of the total lipid weight and is formed by only two components, both sulfatides. Total cholesterol makes up 36% of the total neutral lipid fraction of which cholesterol esters constitute 6%. Glycoproteins are also found to be present in the fraction.     

一种改进的鱼类线粒体DNA的快速制备方法

线粒体DNA(mitOChondrialDNA:mtDNA)是双链环状核外遗传物质,以其分子量小,易于分离,突变率高,进化速度快和母系遗传等特性,已广泛应用于分类学,种系鉴定,种群遗传学,系统发育和进化研究.       

ISOLATION AND PARTIAL CHARACTERIZATION OF MACRO- AND MICRONUCLEI FROM PARAMECIUM AURELIA

A method was developed for the isolation of macro- and micronuclei from Paramecium aurelia. This method utilized ionic and nonionic detergents to rupture the intact cells, calcium ions and spermidine were employed to protect the nuclei, and the nuclei were purified by centrifugation. Macronuclei consisted of 22% DNA, 10% RNA, and 68% protein. Micronuclei were composed of 9% DNA, 11% RNA, and 80% protein. DNA from both macro- and micronuclei had a density of 1.687 g/cc in CsCl and 1.417 g/cc in Cs₂SO₄. These values corresponded to G + C content of about 23%. The RNA of macronuclei was examined by gel electrophoresis, and two high molecular weight species were identified having molecular Weights of 1.3 x 10⁶ and 2.8 x 10⁶ daltons. Three syngens were studied, and in each case the conditions for isolation of the nuclei were the same and no differences were observed in the properties of the nuclei.     

菠菜叶绿体类囊体膜磷酸酯酶的分离和性质

通过正丁醇抽提、离心和柱层析等方法,从菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅａ Ｌ．）叶绿体类囊体膜片分离出磷酸酯酶,此酶水解磷酸单酯类物质。酶活性的最适ｐＨ 在７．０ 以下,属酸性磷酸酯酶。反应温度增至６０℃时反应速度达最高,具有高温酶的特性。ＡＴＰ和无机磷盐均抑制此酶活性。经ＳＤＳ－聚丙烯酰胺凝胶电泳表明,此酶制剂出现两条主要的蛋白带     

柱状田头菇(茶薪菇)金属硫蛋白的分离纯化与特性研究

应用快速灌注色谱系统首次从经Cd2+诱导的柱状田头菇(茶薪菇)Agrocybecylindracea(DC.:Fr:)R.Maoire菌丝体中分离得到一种镉结合蛋白。通过SephadexG-75凝胶过滤层析,原子吸收光谱分析(AAS),巯基含量测定及紫外吸收光谱分析表明这种镉结合蛋白具有金属硫蛋白(metallothionein,MT)的理化性质:即分子量为6kDa、每分子MT含18个半胱氨酸残基并结合7个镉原子、具有镉硫金属簇的特征紫外吸收光谱,初步鉴定为茶薪菇Cd-MT。     

ISOLATION AND CHARACTERIZATION OF GLUCOCEREBROSIDES FROM ADULT RAT BRAIN1

Abstract— By chromatography on borate-coated silicic acid, glucocerebrosides, galactocerebrosides, sulfatides and sphingomyelins from brain tissue could be efficiently separated. Adult rat brain was found to contain 54.1 ± 1.5 nmol of glucocerebrosides per gram fresh weight. Ninety percent of the glucocere-broside fatty acids were palmitate, stearate and oleate; fatty acids with chain lengths above C₂₀ were virtually absent. No hydroxy fatty acids were found. The long chain bases of adult rat brain glucocerebrosides consisted of 74.6% C₁₈-sphingosine, 24.4% C₁₈-sphinganine and 1.1% C₂₀-sphingosine. These results are compared to those obtained from glucocerebrosides from immature rat brains (Abe & Norton , 1974) and discussed in respect to changes occurring during brain development.     

ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF BRUSH BORDERS FROM RABBIT KIDNEY

A technique for the isolation of intact brush borders from rabbit renal cortex was evaluated. The procedure was monitored by phase and electron microscopy and marker enzymes, i.e. ATP:NMN adenylyl transferase, nuclear; cytochrome oxidase, mitochondrial; β-glucuronidase, lysosomal; and glucose-6-Pase, microsomal; and indicated an essentially pure preparation of brush borders. The disaccharidase, trehalase, previously reported in renal tubules, was localized uniquely in brush borders. Maltase was also found; the specific activities of the two enzymes in the brush borders were increased 10- to 20-fold. Other disaccharidases, such as sucrase, isomaltase, lactase, and cellobiase, were absent. It is suggested that trehalase and maltase are appropriate candidates for marker enzymes of the renal brush border. Isolated brush borders possessed a ouabain-sensitive (Na⁺ + K⁺) ATPase, an oligomycin-insensitive Mg⁺⁺ ATPase, and a Ca⁺⁺-activated ATPase. Alkaline phosphatases, dephosphorylating β-glycero-P, and trehalose-6-P were also present. The specific activities of these enzymes were increased three-to-five fold in the brush-border preparations; however, activities were found in other subcellular fractions of the renal cortex. Hexokinase, although evident in the isolated brush border, was found prominently associated with other membranous fractions. Phosphoglucomutase and UDPG pyrophosphorylase were localized in the soluble fraction of the renal cortex.     

海芋胰蛋白酶抑制剂的分离纯化及性质研究

利用亲和层析和分子筛凝胶过滤等技术,从海芋根茎中分离纯化到一种胰蛋白酶抑制剂,简称ＡＭＴＩ。经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ鉴定均显示单一条带,经ＳＤＳ－ＰＡＧＥ测定,其分子量为２２０００,经等电聚焦（ＩＥＦ）测定,其等电点为６．２。根据对胰蛋白酶的抑制比可知该抑制剂为单头抑制剂,其抑制活性在６０℃和ｐＨ５￣１１范围内保持稳定。     

类产碱假单胞菌杀虫物质的分离纯化和鉴定

类产碱假单胞菌是一株昆虫病原菌,该菌对草地蝗虫、竹蝗等具有良好的致死作用,经鉴定,该菌对蝗虫具有毒杀作用的物质为其代谢分泌到胞外的一种蛋白质。类产碱假单胞菌培养物经硫酸按沉淀,SephadexG-100凝胶过滤及DEAE-SephadexA-50阴离子交换柱层析,纯化获得的杀虫蛋白只含一种亚基,分子量25100,等电点5．16,含17种氨基酸,其中谷氨酸含量最高,胱氨酸含量最低,最大吸收峰为278．3nm。     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.     

ISOLATION AND FLOW CYTOMETRIC CHARACTERIZATION OF PROTOPLASTS FROM MARINE MACROALGAE

Protoplasts were isolated from Ulva rigida C. Agardh (Chlorophyta) and two species of Rhodophyta , Gracilariopsis lemaneiformis ( Bory) Dawson, Acleto et Folvik and Gracilaria tenuistipitata Chang et Xia var . liui with minor modifications (the inclusion of 0.01% agarase in the set of cell-wall-degrading enzymes for the two red algae). Flow cytometric characteristics of freshly isolated protoplasts were determined on a FACScan flow cytometer (FC). The most useful parameters for characterizing protoplasts from marine algae were forward angle light scatter (FSC), orange fluorescence (FL2) and red fluorescence (FL3). Protoplasts from all the species were easily distinguishable when their FSC, FL2, and FL3 signals were combined in the bivariate plots FL3 vs. FSC and FL3 us. FL2. Two alternative techniques to help identify protoplasts from debris in the FC computer screen were developed (for FC without sorting capability). Both techniques were based on the ability of new FCs to record time. The first one was based on the induction of rapid changes of cell volume in response to osmotic stress. Only intact protoplasts responded to changes in the osmotic pressure. The second one was based on the uptake and hydrolysis of fluorescein diacetate by intracellular esterases. Viable protoplasts showed a hyperbolic accumulation of fluorescein with time. Semimaximal fluorescein accumulation was attained in 30.5 ± 9.5 s. Debris was easily recognized since, contrary to protoplasts, it did not show a time-dependent accumulation of fluorescein .