多头绒泡菌细胞核周期的电镜研究

多头绒泡菌Ｐｈｙｓａｒｕｍ ｐｏｌｙｃｅｐｈａｌｕｍ Ｓｃｈｗ的营养生长 没有细胞壁的原生质团（合胞体）,内部众多的细胞核进行着同步的核内有丝分裂,本文电镜下研究了细胞核在有丝分裂周期中的结构变化。有丝分裂前期,染色质经松散改组和集缩形成染色体,核仁由中央移向边缘,并在近核膜处解体;中期核膜不消失,在核内形成纺锤体,核仁解体后的物质呈不规则状散在于核内;有丝分裂后核膜的破裂处重新愈合,染色体解集缩     

siRNA对多头绒泡菌丝氨酸/精氨酸蛋白激酶表达的沉默

SR蛋白激酶（serine/arginine protein-specific kinase,SRPK）是一类特异磷酸化剪接因子SR蛋白的激酶家族,可调节SR蛋白的选择型剪接,影响SR蛋白在核内的分布与定位,在pre-mRNA剪接调控上具有重要的作用。目前对于多头绒泡菌SRPK的功能仍不清楚。为了进一步研究PSRPK的功能,设计可转录小干扰RNA（siRNA）的寡核苷酸序列含U6启动子的载体pSIREN-RetroQ定向连接,构建了真核表达质粒pSIREN-PSRPK-1,pSIREN-PSRPK-2,pSIREN-PSRPK-3,pSIREN-PSRPK-4,pSIREN-PSRPK-5及对照质粒pSIREN-PSRPK-Neg。把编码PSRPK基因的cDNA与红色荧光蛋白表达载体pDsRed-N1重组,构建了表达PSRPK-红色荧光融合蛋白的表达质粒pP-SRPK-DsRed。将真核表达质粒和质粒pPSRPK-DsRed共转染HEK293细胞。转染后72h通过倒置荧光显微镜观察,结果表明,pSIREN-PSRPK-2和pSIREN-PSRPK-5可以有效沉默红色荧光融合蛋白的表达;进一步采用RT-PCR和Northern点杂交分析表明,pSIREN-PSRPK-2和pSIREN-PSRPK-5对红色荧光融合蛋白mRNA表达具有明显的抑制作用与倒置荧光显微镜下观察到的结果一致。这为进一步研究多头绒泡菌SRPK的功能奠定了实验基础。     

多头绒泡菌中动蛋白的免疫化学鉴定

动蛋白(kinesin)是一种微管系统的运动蛋白(motor protein),它能通过水解ATP将化学能转化为机械能,推动微管产生运动.微管系统作为一种主要的细胞骨架存在于所有真核细胞中,它们对于维持细胞形态,细胞的分裂,染色体的运动及细胞内的物质运输起着重要作用.细胞质力蛋白(dynein)和动蛋白是公认的推动这类运动的运动蛋白.自从1985年Vole首次在鱿鱼大轴突(squidgiant axon)中发现动蛋白以来,人们先后在许多种动物细胞中发现有动蛋白存在,甚至在低等真核生物棘状变形虫,盘基网柄茵和高等植物烟草花粉管中发现有动蛋白的存在.研究结果表明,动蛋白参与了真核细胞中的许多重要生命活动,如细胞中的细胞器及囊泡的运动,染色体排裂和分离等运动.动蛋白很可能是普通存在于所有真核细胞中的一种运动蛋白.多头绒泡菌(Physarum poly-cephalum)属于粘菌纲(Myxomycetes)的一种低等真核生物,它表现出许多显著的细胞运动特征如原生质团迁移,细胞质的穿梭运动(shuttle streaming)等,是研究非肌细胞运动和收缩蛋白的经典材料.在多头绒泡菌胞质中也具有微管系统,它们构成其纺锤丝等,参与染色体的运动及其它胞质运动,但至今国内外尚无人证明其中有与微管作用的运动蛋白——动蛋白的存在,作者利用抗牛脑动蛋白的单克隆抗体,     

多头绒泡菌染色体构建过程的形态学研究

以同步核内有丝分裂的多头绒泡菌(Physarum polycephalum)原质团为材料,在有丝分裂周期中连续取材,按常规方法制备超薄切片,在电镜下研究了染色体形态构建的整个过程。有丝分裂前期,首先是G_2期凝集的染色质块逐渐解集缩成为松散状,染色质在松散的同时逐渐改组成直径为80～150nm的松散染色线结构。接着是在松散的染色线上形成一些电子密度高的集缩区,随着集缩区的增多和扩展,染色线缩短变粗,最后形成直径300～350nm的染色体。上述两个过程各需30min左右。与上述过程同时发生的是,核仁由中央位置逐渐移向边缘,前期50min左右时在近核膜处呈团块状解体。染色体形态构建的整个过程约需1h,可分为染色质的松散改组和集缩两个连续的步骤,25～30nm染色质纤维是这一过程中能分辨的最细的形态单位。     

多头绒泡菌肌球蛋白的纯化及性质的研究

从多头绒泡菌中纯化了肌球蛋白，并对其亚基组成及ＡＴＰ酶性质进行了研究。该肌球蛋白是由一种重链（２２５ｋＤ）和两种轻链（２０ｋＤ，１７．５ｋＤ）组成的大分子，其亚基之比为ＨＣ：ＬＣ１：ＬＣ２＝２：４：２。兔肌Ｆ－肌动蛋白能较大激活粘菌肌球蛋白ＡＴＰ酶活性，Ｃａ＾２＋离子也能提高其活性，Ｍｇ＾２＋离子无明显影响，钒酸盐，碘乙酸，对氯汞苯甲酸对其ＡＴＰ酶活性有显著抑制作用。     

多头绒泡菌有丝分裂后细胞核重建过程的形态学研究

以进行自然同步核内有丝分裂的多头绒孢菌（Ｐｈｙｓａｒｕｍｐｏｌｙｃｅｐｈａｌｍ）原生质团为材料,应用常规制片和整体银染后制片的电镜技术研究了有丝分裂后细胞核的形态构建过程,形成新核仁的前体物质在有丝分裂中期时莠在染色体区域的周围,末期时与染色体组一起到达两极,子细胞核刚形成时核仁物质与染色质混合,以后核仁物质相互汇合并同染色质逐渐分开,最后形成一个大核仁,染色质在有丝分裂后期开始解集缩,到两极后在     

多头绒泡菌核仁骨架的研究

从多头绒泡菌（Ｐｈｙｓａｒｕｍ　ｐｏｌｙｃｅｐｈａｌｕｍ　Ｓｃｈｗ）间期细胞核中分离出核仁,用ＤＮａｓｅ　Ｉ、０．２５ｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４和２ｍｏｌ／ＬＮａＣｌ去除核仁ＤＮＡ和大部分蛋白质,得到核仁骨架。核仁骨架是直径１０３０ｎｍ的纤维组成的网络结构,含有约２０种多肽,其中包括与肌动蛋白电泳迁移率相当的４３ｋＤ左右的多肽。免疫荧光检测结果表明,核仁骨架能与肌动蛋白抗体结合而发出明亮的荧光。免疫斑点印迹结果进一步证实,核仁骨架的蛋白质成分中存在肌动蛋白。免疫电镜结果指出,代表肌动蛋白的金颗粒分布在整个核仁中。     

多头绒泡菌肌球蛋白的纯化及性质的研究

从多头绒泡菌中纯化了肌球蛋白,并对其亚基组成及ＡＴＰ酶性质进行了研究。该肌球蛋白是由一种重链（２２５ｋＤ）和两种轻链（２０ｋＤ,１７．５ｋＤ）组成的大分子,其亚基之比为ＨＣ：ＬＣ１：ＬＣ２＝２：４：２。兔肌Ｆ－肌动蛋白能较大激活粘菌肌球蛋白ＡＴＰ酶活性,Ｃａ~（２＋）离子也能提高其活性,Ｍｇ~（２＋）离子无明显影响。钒酸盐,碘乙酸,对氯汞苯甲酸对其ＡＴＰ酶活性有显著抑制作用。     

多头绒泡菌银染核仁周期的电镜研究

以同步化培养的多头绒泡菌（Physarum poldycephalum Schw.)原生质团为材料,应用整体银染技术,电镜下研究了核仁在细胞周期中的超微结构变化。结果变化：核仁成熟时比较大,位于细胞核中央,核仁内可区分出纤维中心、密集纤维成分和颗粒成分等。前期时,核仁向边缘移动,前期末在近核膜处解体,解体的核仁物质主要呈团块状散开。中期时,解体的核仁物质位于细胞核中央染色体区域的周围,染色体上没有特异的银染区域,染色体周边也看不到银染的“鞘”状结构,但在染色体中可见一些散在的银染大颗粒。末期时,核仁物质与染色体一起到达两极,在子细胞核中与正在解集缩的染色质共存一起,以后核仁物质逐渐汇合并与染色质分开。大约在有丝分裂结束120min后,在细胞核中形成一候 中央位置的大核仁,结果提示,低等真核生物的核仁结构和周期变化与高等真核生物的不完全相同。     

多头绒泡菌细胞核周期的电镜研究

多头绒泡菌PhysarumpolycophalumSchw的营养生长阶段为没有细胞壁的原生质团（合胞体）,内部众多的细胞核进行着同步的核内有丝分裂,本文电镜下研究了细胞核在有丝分裂周期中的结构变化。有丝分裂前期,染色质经松散改组和集缩形成染色体,核仁由中央移向边缘,并在近核膜处解体;中期核膜不消失,在核内形成纺锤体,核仁解体后的物质是不规则状散在于核内;有丝分裂后核膜的破裂处重新愈合,染色体解集缩成染色质,分散的核仁物质逐渐合并形成新的核仁。     

Studies on microplasmodia of Physarum polycephalum

Summary Fluorescently labeled actin (TRITC-G-actin) and heavy meromyosin (TRITC-HMM) derived from skeletal muscle and injected into microplasmodia of the acellular slime mold Physarum polycephalum were used to analyze the function of a cortical and fibrillar actin system in living specimens. The plasma membrane-attached cortical system can be labeled with TRITC-G-actin as well as with TRITC-HMM and visualized as a continuous sheath along the entire cell surface. Long-term experiments over time periods of several hours in conjunction with digital grey-value evaluations revealed that changes in the intensity of the fluorescent signal, as caused by alternative contraction and relaxation cycles of the cortical system, are distinctly correlated with periodic changes in the volume and shuttle streaming activity of the microplasmodia. The fibrillar actin system extending through the cytoplasmic matrix can be labeled only with TRITC-HMM. Formation and disappearance of fibrils were found to take place during relaxation and contraction of the cortical system, respectively. Results of the present paper indicate that the cortical actin system is mainly involved in motive force generation for alterations in cell surface morphology and locomotion activity, whereas the fibrillar actin system rather appears to maintain the mechanical stability of microplasmodia.Abbreviations ATP adenosine-5'-triphosphate - BSA bovine serum 'albumin - DTE 1,4-dithioerythrit - EGTA ethyleneglycol-bis-(-amino-ethylether)-N,N,N,N,-tetraacetic acid - HMM heavy meromyosin - PIPES l,4-piperazine-N,N-bis-(2-ethanesulfonic acid) - Rh rhodamine - TRIS Tris-(hydroxylmethyl)-aminomethane - TRITC tetramethyl rhodamine isothiocyanate     

Effects of medium composition on the growth and lipid production of microplasmodia of Physarum polycephalum

Physarum polycephalum is a plasmodial slime mold. One of the trophic stages in the life cycle of this organism is a plasmodium. In submerged culture, plasmodia are fragmented into microplasmodia. The latter both lack cell walls and are capable of rapid growth. There has been limited information on the effects of medium composition on the growth and lipid accumulation of microplasmodia. In this study, optimization of medium components by response surface methodology showed that tryptone and yeast extract concentrations had the most significant effects on lipid and biomass production; significant synergistic interactions between glucose and tryptone concentration on these responses were also recorded. The optimal medium was composed of 20 g/L of glucose, 6.59 g/L of tryptone, and 3.0 g/L of yeast extract. This medium yielded 13.86 g/L of dry biomass and 1.97 g/L of lipids. These amounts are threefold higher than those of the American Type Culture Collection (ATCC) medium. In addition, biomass and lipid production reached maximal values between only 4 and 5 days. Fatty acid compositions analysis by gas chromatography-mass spectrometer (GC–MS) revealed that P. polycephalum lipids consisted mainly of oleic acid (40.5%), linoleic acid (10%), and octadecynoic (15.8%). This is the first report on the fatty acid composition of P. polycephalum microplasmodia. These results suggest that the biomass of microplasmodia could be used as a source of material for direct conversion into biodiesel because of the absence of cell walls or it could also be used as a supplemental source of beneficial fatty acids for humans, albeit with some further evaluation needed.     

Studies on microplasmodia of Physarum polycephalum

Summary Fragments excised from front regions of thinspread Physarum plasmodia were used to examine a possible correlation between the periodical dynamic activity of such specimens and the spatial organization of actin fibrils. Under isotonic conditions, symmetrical contractions and relaxations of the entire fragment alternate with a period of 1–4 min, whereas under isometric conditions local contractions and relaxations occur simultaneously in different regions of the same specimen. Rapid fixation and phalloidin-staining at distinct stages of the contraction-relaxation cycle demonstrates the permanent existence of cytoplasmic actin fibrils under both isometric and isotonic conditions. During the transition from relaxation to contraction the fibrils shorten in length from 25.5 m to 21.0 m and increase in density from 1.2 fibrils/1000 m² to 2.3 fibrils/1000 m². The present results demonstrate that actin fibrils in Physarum plasmodia are not completely decomposed and reformed every contraction-relaxation cycle.Series Studies on microplasmodia of Physarum polycephalum VIII     

Revertants of selfing (gad) mutants in Physarum polycephalum

The conversion of the uninucleate amoebal form of Physarum polycephalum to the multi-nucleate plasmodial form is under the control of a genetic region which contains matA (or mt), a determinant of mating specificity. The region is the site of most gad mutations, which give amoebae the ability to produce plasmodia in clones without mating (ie, to self). In the present study, nonselfing revertants were isolated from two matA2-derived gad mutants and two matA3-derived gad mutants. Some revertants were found to have regained exactly, or nearly, the same phenotype as the original matA2 or matA3 strain. Others expressed new mating types, having gained the ability to mate with strains of the parental matA type. The results are compatible with a model in which new mating types arise from forward mutations (gad) and back mutations (npf or no plasmodium formation) occurring successively in a single gene, matA.     

细胞松弛素B对多头绒泡菌有丝分裂的影响

将细胞松弛素Ｂ（Ｃｙｔｏｃｈａｌａｓｉｎ Ｂ,ＣＢ）注入同步化的多头绒泡菌原质团,在光镜和电镜下跟踪观察有有丝分裂进程,发现多头绒泡菌进入有丝分裂的时间推迟,与未经ＣＢ处理的样品相比,在Ｓ期注入ＣＢ的样品进入有丝分裂的时间推迟３５ｍｉｎ,在Ｇ２早期注入ＣＢ的样品则推迟２０ｍｉｎ;在Ｇ２中期注入的推迟４５ｍｉｎ;在Ｇ２晚期注入的推迟６０ｍｉｎ,说明抑制肌动蛋白的功能则使有丝分裂受到明显影响。ＣＢ处理     

Food deprivation is not a prerequisite for the amoebal to plasmodial transition in Physarum polycephalum

The effect of food supply on the onset of asexual and sexual plasmodium formation in Physarum polycephalum was studied. Asexual differentiation occurs readily in amoebae carrying the matAh mating type allele. The density at which these amoebae begin to differentiate is influenced by the ind locus, which controls the production of a diffusible inducer. The alleles ind-1 and ind-2 are known. Strains carring the ind-1 allele begin plasmodium formation at a low amoebal density (rapid differentiation), while strains carring the ind-2 allele differentiate at a higher amoebal density (slow differentiation). The onset of differentiation is characteristic of the strain and did not change with a 20-fold variation in the number of food bacteria available. Sexual differentiation occurs between compatible amoebal strains. For a given pair of amoebal strains the onset of plasmodium formation occurs at a characteristic cell density that is determined by the genetic backgrounds of the strains. The ind locus is one of the genes that influences this cell density. Plasmodia are formed at a lower cell density in crosses involving compatible amoebae carrying the ind-1 allele than they are in crosses with strains carrying the ind-2 allele. As was found for asexual differentiation, an approximate 20-fold variation in the food supply did not affect the initiation of sexual plasmodium formation. These results suggest that in most cases starvation does not trigger the differentiation of amoebae into plasmodia. The time of onset of plasmodium formation is determined largely by genetic factors.     

Identification of three genes expressed primarily during development in Physarum polycephalum

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation. Received: 21 June 1999 / Accepted: 17 August 1999