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     

多头绒泡菌间期细胞核和中期染色体中的银染蛋白分析

电镜原位观察结合图象分析研究了多头绒泡菌Physarum polycephalum Schw间期细胞核和中期染色体中银染蛋白的形状,大小和分布。结果看到,银染蛋白扩要呈颗粒状存在于间期细胞核和中期染色体中。银粒的大小不一,分布不均匀。间期细胞核中存在侈多直径在5－15nm的银粒,其中10nm以上的较大银粒主要分布于核仁,集缩染色质和核基质部分10nm以上银粒不多,中期细胞核内10nm以上的较大银粒主要分布于染色体中。染色体中除含有一些较大银粒外,多数银粒的直径为5－10nm。本文结果提示,构成染色体骨架的嗜银蛋白可能来自间期细胞核的染色质,核基质和核仁。     

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。     

多头绒泡菌SC35类蛋白的免疫电镜研究

经抗SC35单克隆抗体标记后,在电子显微镜下观察到多头绒泡菌S、G2、前期、中期和后末期细胞核中存在大量金颗粒,说明多头绒泡菌细胞核含有SC35类蛋白。在G2期和前期时,SC35类蛋白主要分布在细胞核的核仁区域和非核仁区域的染色质间区域;中期和后-末期时,SC35类蛋白主要分布在细胞核内染色体间区域;说明染色质(体)间区域和核仁区域是富含SC35类蛋白的区域。对核仁的进一步观察指出,在核仁中金颗粒主要分布在DFC,FC中的金颗粒很少,说明在核仁中SC35类蛋白主要存在于DFC组分中。     

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。     

类Cyclin A蛋白在多头绒泡菌细胞周期中的定位研究

采用免疫电镜技术对多头绒泡菌(Physarum polycephalum)是否含有类CyclinA蛋白以及该蛋白在有丝分裂周期各时相的定位进行了研究;并以抗CyclinA抗体封闭细胞内源类CyclinA蛋白的方法,探讨类CyclinA蛋白在多头绒泡菌细胞周期中的作用。免疫电镜结果表明,经抗CyclinA抗体标记的实验组细胞中的金颗粒密度明显高于对照组,说明多头绒泡菌细胞中含有类CyclinA蛋白。实验组样品中,细胞核的金颗粒密度很高,而细胞质的金颗粒密度与对照组的相仿,说明多头绒泡菌细胞中的类CyclinA蛋白是核蛋白。细胞核的金颗粒密度在S期最高,G2期的次之,早中期时明显降低,中期和中期以后与对照组的相近。这种金颗粒密度的变化反映了类CyclinA蛋白在细胞周期中的含量变化。以抗CyclinA抗体分别处理S期和G2期的多头绒泡菌细胞,处理后的细胞分别停滞在原来的时相,细胞核形态变得不规则,核内有空洞现象。处于有丝分裂前期的多头绒泡菌细胞经抗CyclinA抗体处理后,细胞核出现畸变。抗体处理结果说明类CyclinA蛋白是参与多头绒泡菌细胞周期多个转换过程调控的种重要蛋白,主要在S期／G2期和G2期／M期的转换以及走出有丝分裂期的进程中发挥作用。     

间期细胞银染活性核仁形成区的电镜观察方法

本文报告改进建立的透射电镜砚察间期细胞银染活性核仁形成区的方法简便易行,能在同一个细胞中从亚细胞和分子水平上清楚地显示银染蛋白、活性rDNA和rRNA三者间的关系。     

多头绒泡菌间期细胞核和中期染色体中的银染蛋白分析*

电镜原位观察结合图象分析研究了多头绒泡菌Physarum polycephalum Schw间期细胞核和中期染色体中银染蛋白的形状、大小和分布。结果看到,银染蛋白主要呈颗粒状存在于间期细胞核和中期染色体中。银粒的大小不一,分布不均匀。间期细胞核中存在众多直径在5~15nm的银粒,其中10nm以上的较大银粒主要分布于核仁,集缩染色质和核基质部分10nm以上银粒不多。中期细胞核内10nm以上的较大银粒主要分布于染色体中。染色体中除含有一些较大银粒外,多数银粒的直径为5~10nm。本文结果提示,构成染色体骨架的嗜银蛋白可能来自间期细胞核的染色质、核基质和核仁。     

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     

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     

多头绒泡菌类cyclinB1蛋白在细胞周期中的变化

以自然同步化的多头绒泡菌（Physarum polycephalumL.)为材料,经抗cyclinB1抗体的免疫印迹和免疫电镜实验观察结果表明,多头绒泡菌中含有类cyclinB1蛋白,该蛋白的含量和细胞内位置在细胞周期进程中存在着动态变化。类cyclinB1蛋白在S期开始合成并在细胞质中积累,G2晚期开始进入细胞核,该蛋白在细胞质和细胞核中含量逐渐增加。有丝分裂中期时达最大值。后末期时骤然消失,在G2晚期到有丝分裂中期期间,类cyclinB1蛋白既是细胞核蛋白又是细胞质蛋白,细胞质是类cyclinB1蛋白的主要存在区域,细胞核中的类cyclinB1蛋白主要结合于染色体和核仁区域。     

多头绒泡菌间期细胞核中RNA的转录状况

利用BrUTP免疫标记技术,研究了多头绒泡菌（Physarum polycephalum Sclhw.）间期细胞核中RNA的转录状况。结果表明：在整修间期核仁中的rRNA都在活跃转录;核质中hnRNA的转录呈逐渐上升趋势,早S期转录水平很低,晚S期转录活性升高1倍,G2期转录达到最高水平;整修间期核质中RNA的转录水平增加了5－6倍。     

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     

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.     

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.     

多头绒泡菌PSCL32.5蛋白的性质及其含量在细胞周期中的变化

用免疫印迹技术检测到低等真核生物多头绒泡菌中含有两种与HeLa细胞SC35单克隆抗体反应的蛋白,其分子量分别为32．5kD和82．5kD,将其命名为PSCL32．5和PSCL82．5。用SDS—PAGE技术对PSCL32．5蛋白进行了纯化,用等电聚焦方法确定PSCL32．5蛋白的等电点为6．19。应用免疫印迹技术检测多头绒泡菌细胞周期不同时相的PSCL32．5含量,发现该蛋白的含量在细胞周期中是变化的,在S早期时含量最低,从S期到G2期含量逐步增高,G2期后期含量最高。     

A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein,ASF/SF2

A 1591-bp cDNA of a serine-rich protein kinase （SRPK）-Iike protein has been identified in Physarum polycephalum （GenBank accession No. DQ140379）. The cDNA contains two repeat sequences at bp 1-153 and bp 395-547. The encoding sequence is 56% homologous to human SRPK1 and is named Physarum SRPK （PSRPK）. Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains （CDs）. However, divergent motifs between the N-terminal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphorylation assay to investigate the phosphorylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coil cells. Autoradiographic examination showed that both recombinant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could phosphorylate ASF/SF2 and a truncated form of ASF/SF2 that contains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and function of PSRPK demonstrates that it is a new member of the SRPK family.