双壳贝类染色体标本制备技术的优化

本研究以牡蛎、咬齿牡蛎鳃细胞为材料,采用PHA处理法、低温同步化法及活体去壳法进行预处理,制作成染色体标本;以马氏珠母贝、企鹅珍珠贝担轮幼虫为材料,采用PHA促繁殖获取胚胎法,制作胚胎染色体标本.对不同时间不同处理方式获得牡蛎染色体分裂相比例、PHA促繁殖获取胚胎法获得马氏珠母贝染色体分裂相比例进行统计.结果发现,PHA处理法在24 h时能获取最多分裂相(3.50‰),低温同步化法在72 h时能获得最多分裂相(2.20‰),活体去壳法始终保持较高分裂相比例(3.60‰),PHA促繁殖获取胚胎法获得最大分裂相比例(10.00‰).4种方法都能获得理想的中期分裂相数目.其中,低温同步化法缺点是温度难控制、预处理时间长;活体去壳法易导致贝类死亡;PHA促受精获取胚胎法缺点是胚胎的获取受繁殖季节限制.PHA处理法预处理时间短、获取分裂相数目多,无疑是贝类成体染色体制备的最好的方法.同时,PHA也为贝类人工繁殖提供了一种更为有效的手段.     

Chromosome Aberrations and Ageing Root Meristems

Changes in mitotic activity and frequency of spontaneous chromosomeaberrations have been determined for primary roots of Zea muys,Pisum sativum, Vicia faba var. green Windsor and var. minor,and Allium cepa. Mitotic activity was found to decline withage in all root apices. In the primary root apices immediately after germination, therewas a high frequency of spontaneous chromosome aberrations whichdeclined with time after germination. However, In the case ofV. faba var. green Windsor, Z mays and P. sativum there wasa return to the high levels of spontaneous chromosome aberrationon ageing of the root apex. root meristems, mitotic activity, chromosome aberrations, ageing     

The MRN-CtIP Pathway Is Required for Metaphase Chromosome Alignment

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Highlights? The MRN-CtIP pathway is required for metaphase chromosome alignment in egg extracts ? MRN inhibition interferes with spindle assembly around DNA-coated beads ? MRN inhibition in cells causes a metaphase delay and disrupts the RanGTP gradient ? The MRN complex regulates the stable association of RCC1 to chromatin     

Localization of Nucleic Acid Synthesis in Root Meristems

Adenine-8-C1¹⁴ was supplied to roots of Vicia faba and Alliumascalonicum and its incorporation into DNA was studied fromautoradiographs of hydrolysed sections. These roots have a quiescentcentre to the meristem where the cells do not synthesize DNAand probably, therefore, play no part in the construction ofthe root. The boundary between the quiescent centre and thecentral cap initials is clearly denned and this suggests thatthere is as little cell interchange between the histogens asthere is in roots with visibly discrete histogens.     

Haspin: A Mitotic Histone Kinase Required for Metaphase Chromosome Alignment

The fidelity of chromosome segregation during cell division is critical to maintaingenomic stability and to prevent cancer and birth defects. A key set of kinases that regulates thisprocess has been identified and characterized over the last few years, including the Aurora, Poloand Nek families. Recently we proposed that a little-studied kinase known as haspin is a newmember of this important group. During mitosis haspin is phosphorylated, associates with thechromosomes, centrosomes and spindle, and is responsible for phosphorylation of histone H3 atthreonine-3. Depletion of haspin using RNA interference prevents normal alignment ofchromosomes at metaphase, suggesting that haspin plays a crucial role in chromosomesegregation. Here we discuss possible mechanisms of haspin action and the function of histonephosphorylation in mitosis. We also outline some of the questions raised by these new findingsand consider what role haspin might play in cancer.     

Somatic Chromosome Counts from Leaf Meristems in the Tribe Triticeae

The Feulgen procedure was applied to chromosome preparations of leaf meristems from growing plants of wheat, barley, and wheat-wheatgrass hybrids. Leaf primordia from the base of secondary tillers were pretreated in cold water (overnight, 2 C), fixed in glacial acetic acid (20 min, 2 C), hydrolyzed in 1 N HCl (14 min), stained in leuco-basic fuchsin (about 15 min) and squashed in 1% acetocarmine. The chromosome spreads from leaf meristems were generally superior to those of the root meristems of the same plant. Mitotic index in leaf meristems was higher than in root meristems in some species. The method appears useful for counting the chromosome number of growing plants, detecting chimeras, and verifying root tip chromosome counts.     

Cytologic and Autoradiographic Studies of the Micronucleus at Meiotic Prophase in Tetrahymena pyriformis

Micronuclear changes of variety 1 of Tetrahymena pyriformis during meiotic prophase have been observed by the light microscope. Morphologic changes in the micronucleus are divided into 6 stages. In stage I, chromatin begins to polarize; in stage II, the micronucleus becomes spindle shaped; and in stage III, one end of the micronucleus protrudes to form a “neck.” In stage IV, where the micronucleus elongates to maximal length, the whole micronucleus consists of 2 chromatin threads pairing longitudinally. One thread probably contains one genome. In stage V, the elongated thread becomes shorter and thicker. Finally, in stage VI, separate chromosomes appear and enter into metaphase. To discover the role of the elongation of the micronucleus, called crescent formation, autoradiographic analysis of RNA and DNA synthesis were undertaken using [³H]uridine and [³H]thymidine. Pulse label and chase experiments show that the crescent in stages II and III is actively synthesizing RNA. Though no remarkable DNA synthesis was observed during meiosis, a small amount of DNA synthesis occurred during the 1st and 2nd prezygotic divisions.     

Improved Methods for Permanent Chromosome Preparations: Cellophane Squashes and Citric Acid Dissociation

Normally, squash preparations from prestained acetic acid softened or HCl macerated tissues are made by pressing the tissue under a coverglass (e.g. Walker 1973). When permanent slides are wanted the coverglass has to be removed sooner or later, which is only possible by hardening the squashed tissue by freezing, for example in carbon dioxide snow, or by slow diffusion of fixatives into the space between the slide and the coverglass. These methods are either expensive or time-consuming, and upon removal of the coverglass, many cells and chromosomes either are lost or are poorly preserved.     

Evidence for a Role of CLIP-170 in the Establishment of Metaphase Chromosome Alignment

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.Microtubules (MTs)¹ in vertebrate somatic cells are involved in intracellular transport and distribution of membranous organelles. Fundamental to this role are their tightly controlled, polarized organization, and unusual dynamic properties (Hirokawa, 1994) and their interaction with a complex set of MT-based motor proteins (Hirokawa, 1996; Sheetz, 1996; Goodson et al., 1997). During mitosis, they contribute to the motility of centrosomes, the construction of spindle poles (Karsenti et al., 1996; Merdes and Cleveland, 1997), and the dynamic movements of kinetochores (Rieder and Salmon, 1994) and chromosome arms (Barton and Goldstein, 1996; Vernos and Karsenti, 1996). The motor protein cytoplasmic dynein, drives the transport toward MT minus-ends of a variety of subcellular organelles (Schnapp and Reese, 1989; Schroer et al., 1989; Holzbaur and Vallee, 1994). Dynactin is a molecular complex originally identified as being essential for dynein-mediated movement of salt-washed vesicles in vitro (reviewed in Schroer, 1996; Schroer and Sheetz, 1991). Genetic studies in fungi, yeast, and flies have shown that the two complexes function together to drive nuclear migration, spindle and nuclear positioning and to permit proper neuronal development (Eshel et al., 1993; Clark and Meyer, 1994; Muhua et al., 1994; Plamann et al., 1994; McGrail et al., 1995; Karsenti et al., 1996). Biochemical studies suggest a direct interaction between certain subunits of dynein and dynactin (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995). In vivo, the two molecules may bind one another transiently, since they have not been isolated as a stable complex.There is good evidence indicating that the dynein/dynactin complex, together with other motors (Eg5, and a minus-end oriented kinesin-related protein) and a structural protein (NuMa), drive the focusing of free microtubule ends into mitotic spindle poles (Merdes and Cleveland, 1997; Waters and Salmon, 1997)_. A trimolecular complex composed of NuMa and dynein/dynactin may be crucial in this process in both acentriolar (Merdes et al., 1996), and centriolar spindles (Gaglio et al., 1997). A number of findings also indicate that the combined actions of dynein and dynactin at the kinetochore contribute to chromosome alignment in vertebrate somatic cells. First, the initial interaction between polar spindle MTs and kinetochores seems to involve a tangential capture event (Merdes and De Mey, 1990; Rieder and Alexander, 1990) which is followed by a poleward gliding along the surface lattice of the MT (Hayden et al., 1990). Both in vivo and in vitro (Hyman and Mitchison, 1991) this gliding movement appears similar to the dynein-mediated retrograde transport of vesicular organelles along MTs. Consistent with this is the finding that both dynein (Pfarr et al., 1990; Steuer et al., 1990) and its activator, dynactin (Echeverri et al., 1996), are present at prometaphase kinetochores. Overexpression of dynamitin, a 50-kD subunit of the dynactin complex, results in the partial disruption of the dynactin complex and in the loss, from kinetochores, of dynein, as well as dynactin. Therefore, it has been proposed that dynactin mediates the association of dynein with kinetochores. Abnormal spindles with poorly focused poles are observed and the cells become arrested in pseudoprometaphase (Echeverri et al., 1996). Despite these findings, rigorous proof for a role of the dynein motor complex in kinetochore motility is still lacking, and its role may differ between lower and higher eucaryotes, and between mitosis and meiosis.CLIP-170 (Rickard and Kreis, 1996) is needed for in vitro binding of endocytic transport vesicles to MTs (Pierre et al., 1992). It is a nonmotor MT-binding protein that accumulates preferentially in the vicinity of MT plus ends and on early endosomes and endocytic transport vesicles in nondividing cells (Rickard and Kreis, 1990; Pierre et al., 1992). Like many MT-binding proteins, CLIP-170 is a homodimer whose NH₂-terminal head domains and COOH-terminal tail domains flank a central α-helical coiled-coil domain. The binding of CLIP-170 to MTs involves a 57–amino acid sequence present twice in the head domain (Pierre et al., 1992) and is regulated by phosphorylation (Rickard and Kreis, 1991). The COOH-terminal domain has been proposed to participate in targeting to endocytic membranes (Pierre et al., 1994). The fact that the latter move predominantly toward microtubule minus ends in a process most likely mediated by cytoplasmic dynein and dynactin (Aniento and Gruenberg, 1995), suggests that CLIP-170 may act in concert with this motor complex, and may be subject to regulated interactions with one or more dynactin or dynein subunits at the vesicle membrane.Here we report that during mitosis, CLIP-170 codistributes with dynein and dynactin at kinetochores, but not spindle poles. Evidence is presented that the COOH-terminal domain of CLIP-170 is responsible for its kinetochore targeting, and that this may be mediated by the complex of dynein and dynactin. The effects on mitotic progression of overexpression of wild type and several deletion mutants of CLIP-170 provide evidence for the involvement of CLIP-170 in kinetochore function early in mitosis. We also present in vivo evidence that neither CLIP-170 nor the complex of dynein and dynactin are required for formation of kinetochore fibers.     

Role of Water in Chromosome Spreading and Swelling Induced by Acetic Acid Treatment: A Ftir Spectroscopy Study

The so called chromosome preparation is a procedure consisting of three strictly connected stages that enables to obtain chromosomes of quality suitable for cytogenetic analysis. Interestingly, experimental evidence strongly suggested that chromosome spreading and swelling (key processes that allow their counting and detailed structural analysis) are induced in the last fixative-evaporation stage by the interaction, mediated by acetic acid, between water from the environmental humidity, and the cytoplasmic matrix and the chromatin. However, since a considerable variation in the quality of chromosome preparations is observed, strongly depending on the environmental conditions in which the procedure takes place, a better comprehension of the mechanisms underlying chromosome preparation is required. To this aim, here we analysed intact lymphocytes before and at each stage of the chromosome preparation protocol by Fourier transform infrared (FTIR) spectroscopy, a technique widely used for the study not only of isolated biomolecules, but also of complex biological systems, such as whole cells. Interestingly, we found that the chromosome preparation protocol induces significant structural changes of cell proteins and DNA, in particular due to the interaction with acetic acid. Moreover, noteworthy, through the monitoring of changes in the water combination band between 2300 and 1800 cm^–1, we provided evidence at molecular level of the crucial role of the bound water to the cytoplasmic matrix and to the chromatin in determining the chromosome spreading and swelling. Our FTIR results, therefore, underline the need to perform the last fixative-evaporation stage in standardized and optimized temperature and relative humidity conditions, thus providing chromosomes of high quality for the cytogenetic analysis that would lead in this way to more reliable results.Key words: Chromosome preparation, chromosome spreading and swelling, DNA conformational transition, FTIR (micro)spectroscopy, protein, DNA hydration     

小鼠中期染色体制备方法探讨

目的:寻找小鼠细胞中期染色体制备过程中理想的取材方法和低渗的最佳时间,以及制片时滴片的最佳高度。方法:根据以往小鼠染色体制备方法的基本步骤,在取材方面进行对比,同时分别设置4个加入低渗液的时间和4个滴片高度进行试验对比。结果:各低渗时间段和各滴片高度所制备的结果存在一定的差异性,制备成功率和良好率最高的低渗时间为25 min,滴片高度为20 cm。结论:取材骨髓比较容易和简洁制备中期染色体,最佳低渗时间为25 min,最佳滴片高度为20 cm。     

Morphogenetic Studies on the Distribution and Activities of Leaf Meristems in Ferns

In very young sporophytes of Dryopteris and Osmunda, the leafprimordia originate very close to the shoot apical cell andshow early differentiation of an apical cell, rapid growth,and an early transition from distal to marginal growth. In successively older primordia of adult Dryopteris, a gradualelaboration in the size of the leaf apical cell takes placeand the greatest size is attained before lateral pinnae beginto be formed. With the formation of lamina, the apical cellgradually decreases in size and is transformed into the marginaltype of meri-stematic cell, when the leaf unrolls. In ferns with a homogeneous marginal meristem, which consistsof a uniform layer of cells with an equal capacity for growth,a simple, entire leaf is formed, e.g. Phyllitis and Platyceriumand where an initially homogeneous marginal meristem becomesheterogeneous, with a consequential differentiation of areasof unequal growth, a lobed or pinnate configuration, as in Blechnumand Lomnaria, or a compound leaf, as in Dryopteris, results. There are some indications of the inception of vascular elementsbeing due to the activity of functioning meristems, the processbeing a basipetal one.     

Rejuvenation of Meiotic Cohesion in Oocytes during Prophase I Is Required for Chiasma Maintenance and Accurate Chromosome Segregation

Chromosome segregation errors in human oocytes are the leading cause of birth defects, and the risk of aneuploid pregnancy increases dramatically as women age. Accurate segregation demands that sister chromatid cohesion remain intact for decades in human oocytes, and gradual loss of the original cohesive linkages established in fetal oocytes is proposed to be a major cause of age-dependent segregation errors. Here we demonstrate that maintenance of meiotic cohesion in Drosophila oocytes during prophase I requires an active rejuvenation program, and provide mechanistic insight into the molecular events that underlie rejuvenation. Gal4/UAS inducible knockdown of the cohesion establishment factor Eco after meiotic S phase, but before oocyte maturation, causes premature loss of meiotic cohesion, resulting in destabilization of chiasmata and subsequent missegregation of recombinant homologs. Reduction of individual cohesin subunits or the cohesin loader Nipped B during prophase I leads to similar defects. These data indicate that loading of newly synthesized replacement cohesin rings by Nipped B and establishment of new cohesive linkages by the acetyltransferase Eco must occur during prophase I to maintain cohesion in oocytes. Moreover, we show that rejuvenation of meiotic cohesion does not depend on the programmed induction of meiotic double strand breaks that occurs during early prophase I, and is therefore mechanistically distinct from the DNA damage cohesion re-establishment pathway identified in G2 vegetative yeast cells. Our work provides the first evidence that new cohesive linkages are established in Drosophila oocytes after meiotic S phase, and that these are required for accurate chromosome segregation. If such a pathway also operates in human oocytes, meiotic cohesion defects may become pronounced in a woman''s thirties, not because the original cohesive linkages finally give out, but because the rejuvenation program can no longer supply new cohesive linkages at the same rate at which they are lost.     

Putative ABC Transporter Responsible for Acetic Acid Resistance in Acetobacter aceti

Two-dimensional gel electrophoretic analysis of the membrane fraction of Acetobacter aceti revealed the presence of several proteins that were produced in response to acetic acid. A 60-kDa protein, named AatA, which was mostly induced by acetic acid, was prepared; aatA was cloned on the basis of its NH₂-terminal amino acid sequence. AatA, consisting of 591 amino acids and containing ATP-binding cassette (ABC) sequences and ABC signature sequences, belonged to the ABC transporter superfamily. The aatA mutation with an insertion of the neomycin resistance gene within the aatA coding region showed reduced resistance to acetic acid, formic acid, propionic acid, and lactic acid, whereas the aatA mutation exerted no effects on resistance to various drugs, growth at low pH (adjusted with HCl), assimilation of acetic acid, or resistance to citric acid. Introduction of plasmid pABC101 containing aatA under the control of the Escherichia coli lac promoter into the aatA mutant restored the defect in acetic acid resistance. In addition, pABC101 conferred acetic acid resistance on E. coli. These findings showed that AatA was a putative ABC transporter conferring acetic acid resistance on the host cell. Southern blot analysis and subsequent nucleotide sequencing predicted the presence of aatA orthologues in a variety of acetic acid bacteria belonging to the genera Acetobacter and Gluconacetobacter. The fermentation with A. aceti containing aatA on a multicopy plasmid resulted in an increase in the final yield of acetic acid.     

Chromosome Studies in Leukemia

This review discusses the significance of chromosomal abnormalities found in leukemia with the bias of belief that these have a primary role or are the mechanism of action of leukemogenic agents. The Philadelphia chromosome (Ph¹) is present in marrow cells examined without culture at any stage of most patients with chronic granulocytic leukemia (CGL). the presence of this chromosome is of diagnostic and prognostic value.Varied chromosomal abnormalities have been found in acute leukemia. Each abnormality, which may be unique, is absent in remission, found again at relapse and is seldom changed by therapy. Abnormalities may be of number of chromosomes (aneuploid) or structural rearrangements resulting in “marker” chromosomes, Ranges of abnormal numbers of chromosomes, when present, usually have related patterns which suggest origin of several cell types from one initial cell. Cells from patients with increased risk of leukemia owing to genetic factors have a high incidence of chromosome breakage and structural rearrangements suggesting a mechanism for production of clones of abnormal, possibly leukemic, cells.     

Mechanism of Acetic Acid Gustatory Repulsion in Drosophila

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Internodal Meristems of Monocotyledons: Further Studies and a General Taxonomic Summary

The occurrence of uninterrupted (UM) and intercalary (IM) meristemsin the internodes of vegetative and/ or reproductive axes isreported for 90 species in 34 familie of monocotyledons. Meristemtype was determined by measuring regions of surface elongationand cell division in developing internodes. Many aquatic andpredominantly tropical families are included. These new dataand previously published information are presented in a summaryof 45 families in 19 orders of monocotyledons (sensu Takhtajan,1969). The distribution of UM's and IM's is about equal amongfamilies and orders. Phylogenetic trends are not apparent inthe distribution. Generally, UM's tend to predominate in vegetativeinternodes, and IM's tend to predominate in reproductive internodes.