植物非整倍体研究进展

非整倍体(aneuploid)是指相对于正常个体(euploid)的染色体组增加、减少一条或若干条染色体的生物个体。由于非整倍体个体存在基因剂量效应的不平衡性(gene-dosage imbalance),非整倍体个体往往会表现严重的表型缺陷(aneuploid syndrom),如发育迟缓,个体矮小,难以繁殖后代等。在人类中,最为典型的例子为导致新生儿智力缺陷的唐氏综合症,由额外的一个21号染色体拷贝(部分拷贝)引起。此外,大多数癌细胞类型表型为严重的非整倍体。在大多情况下,非整倍体对于动物及人类是致命的,而植物对于非整倍体则往往表现出较强的耐受力,特别是在异源多倍体植物中。植物非整倍体对于植物的遗传、育种研究有重要意义,在基因及分子标记的物理位置确定,基因转移,连锁群与染色体的对应关系的确立上具有无可比拟的优势。该文综述了近些年来有关植物非整倍体研究的结果,介绍了非整倍体的几种重要成因和有关非整倍体鉴定手段的变迁,阐述了植物非整倍体对个体表型、基因表达以及表观遗传方面的影响,重点讨论了非整倍体在植物进化、基因组序列测定以及遗传改良方面的潜在作用。同时,探讨了植物非整倍体研究的新思路,以及利用非整倍体促进相关植物遗传改良、育种研究的新方法。     

端粒酶抑制剂对不同非整倍体状态的人结肠癌HCT116细胞增殖及凋亡的影响

该文研究端粒酶在人结肠癌HCT116高非整倍体变异组及低非整倍体变异组细胞中的表达差异及其端粒酶抑制剂3′-叠氮-3′-脱氧胸苷(3′-Azido-3′-deoxythymidine,AZT)对2组细胞增殖及凋亡的影响。取HCT116细胞加入盐酸强力霉素16 h后撤药,称为高非整倍体变异组;另取HCT116细胞不作任何处理,设为对照组,称为低非整倍体变异组;在撤药后第11 d,采用100、250μmol/L的AZT处理2组细胞72 h,并分别设立空白对照组(未加AZT的高非整倍体变异组及低非整倍体变异组)。采用染色体滴定法进行染色体计数。采用Western blot检测MAD2L1、PUMA、BAX、P21、γ-H2AX蛋白质水平。采用荧光定量PCR检测h TERT、PUMA、BAX、NOXA、P21基因表达,端粒酶活性试剂盒检测端粒酶活性,CCK-8法检测细胞生存率。实验结果表明,采用盐酸强力霉素诱导HCT116细胞的非整倍体率可达到77.33%;高非整倍体变异组细胞h TERT基因的表达及端粒酶活性明显高于低非整倍体变异组;加入AZT后,高非整倍体变异组P21、γ-H2AX蛋白质水平上升程度较整倍体明显,低非整倍体变异组PUMA、BAX蛋白质水平上升程度较高非整倍体变异组明显。该研究表明,盐酸强力霉素可以诱导非整倍体形成,高非整倍体变异组的端粒酶活性及h TERT基因表达高于低非整倍体变异组,AZT可以对高非整倍体变异组和低非整倍体变异组细胞产生增殖抑制作用、DNA损伤作用、细胞周期阻滞作用、诱导凋亡作用。     

昆明山海棠根部水抽提物对体外微管蛋白聚合的影响

本研究利用猪脑中分离纯化的微管蛋白聚合和解聚反应,分析了非整倍体诱发剂昆明山海棠根部水抽提物(THH)对微管蛋白聚合状态的影响,从该角度探讨了THH诱发哺乳动物非整倍体的机制。秋水仙素(COL)为本研究的阳性对照物。结果发现THH能显著抑制体外微管蛋白的聚合,该抑制效应呈明显的剂量——效应关系。研究结果与我们以往关于THH为非整倍体诱发剂的实验证据相吻合并进一步提示THH可以抑制微管蛋白聚合作为诱发非整倍体的途径之一。     

激光诱变无核沙田柚的染色体研究

选摘经激光诱变得到的第二代无核(少核)沙田柚叶片,和未经激光处理的对照组进行染色体的对比分析,实验分析发现染色体数目发生了改变,出现了非整倍体。本文对染色体数目进行了统计分析,并讨论了非整倍体与无核化之间的关系。     

小麦端体、单体及其测交后代的细胞学研究

小麦非整倍体研究始于三十年代,E.R.Sears最早开创。他开创的“中国春”小麦单体、缺体、三体和四体等完整系统在小麦的遗传学、系统发育学和育种学方面的应用越来越广泛。现在各国已用各自适用的品种培育出成套单体系统62个。为在小麦遗传育种研究上更好地应用小麦非整倍体材料进行小麦种质改良,导入一些近缘种属间的优良基因,就必须首     

三种有丝分裂抑制剂诱发小鼠骨髓细胞非整倍体的比较研究

以秋水仙素有丝分裂(CM)效应、微核(MN)及染色体畸变(CA)三种体内细胞遗传学指标综合评估了有丝分裂抑制剂(秋水仙素、益康唑及对苯二酚)诱发小鼠骨髓细胞非整倍体的效应。结果表明:秋水仙素是典型的多倍体及非整倍体诱发剂。益康唑对细胞有丝分裂有与秋水仙素相类似的效应,进一步分析表明其在哺乳动物体细胞内无非整倍体诱发效应。对苯二酚在哺乳动物活体实验系统中,可能具有诱发非整倍体及染色体结构畸变的多种遗传毒性。结果提示三种细胞遗传学指标能为非整倍体诱发剂的检出提供依据。     

丙烯酰胺非整倍体诱发效应的荧光原位杂交和CREST染色的研究

本文以小鼠着丝粒次要卫星DNA探针FISH和抗着丝粒CREST染色,研究了可疑的非整倍体毒剂丙烯酰胺(AA)诱导的小鼠NIH3T3细胞微核(MN)的着丝粒组成情况和小鼠骨髓染色体畸变(CA)情况。结果发现AA在100—400μg/ml诱导的MN约52.7％—71.6％为FISH阳性,60.5％—68.2％的MN为CR-EST阳性,两种结果均显示AA具有较强的非整倍体诱发效应。小鼠骨髓CA的FISH表明,AA既能诱导染色体结构畸变,又能诱导非整倍体形成,而以非整倍体诱发效应更为明显。     

昆明山海棠对中国仓鼠V79细胞二酰基甘油含量的影响

本研究以昆明山海棠根部水抽提物(Tripterygium Hypoglaucum(Level)Hutch,THH)处理中国仓鼠V79细胞,通过检测V79细胞C-M细胞频率以及二酰基甘油(1,2-diacylgcerol,DAG)的含量测定,分析了THH诱发非整倍体与细胞醇磷酯信号通路的关系.结果指出THH能在1mg/ml、2mg/ml两个剂量上使V79细胞的DAG含量显著升高(P＜0.001),并明显的提高C-M细胞频率(P＜0.05),提示肌醇酯信号通路是介导THH诱发非整倍体的途径之一.     

植物非整倍体及其遗传

生物物种在长期的进化过程中形成了稳定的遗传体系,都具有整倍的染色体数。但整倍体生物能够自发地或人为地产生非整倍体。植物非整倍体(三体)是在曼陀罗中首先发现的,并且在育种的过程中陆续培育出12种可能的三体。此后,又在烟草和燕麦中发现单体等非整倍体。     

2（4′噻唑）苯丙咪唑对猪脑微管蛋白体外聚合的影响

本研究利用猪脑中分离纯化的微管蛋白聚合和解聚反应,分析了具有争议的非整倍体诱发剂2（4′噻唑）苯丙咪唑（thiabendazole,TBZ）对微管蛋白聚合状态的影响。秋水仙素(colchicine)为本研究的阳性对照物。结果发现2（4′噻唑）苯丙咪唑能显著抑制体外微管蛋白的聚合,并呈明显的剂量效应关系。研究表明,TBZ可能通过抑制微管蛋白聚合来影响染色体正常分离,诱发非整倍体。     

When 2+2=5: the origins and fates of aneuploid and tetraploid cells

Aneuploid cells are frequently observed in human tumors, suggesting that aneuploidy may play an important role in the development of cancer. In this review, I discuss the processes that may give rise to aneuploid cells in normal tissue and in tumors. Aneuploid cells may arise directly from diploid cells through errors in chromosome segregation, as a consequence of incorrect microtubule-kinetochore attachments, or through failure of the spindle checkpoint. A second route to formation of aneuploid cells is through a tetraploid intermediate, where division of tetraploid cells can yield very high rates of chromosome missegregation as a consequence of multipolar spindle formation. Diploid cells may become tetraploid through a variety of mechanisms, including endoreduplication, cell fusion, and cytokinesis failure. Although aneuploid cells may arise from either diploid or tetraploid cells, the fate of the resulting aneuploid cells may be distinct. It is therefore important to understand the different pathways that can give rise to aneuploid cells, and how the varied origins of these cells affect their subsequent ability to survive or proliferate.     

When 2 + 2 = 5: The origins and fates of aneuploid and tetraploid cells

Aneuploid cells are frequently observed in human tumors, suggesting that aneuploidy may play an important role in the development of cancer. In this review, I discuss the processes that may give rise to aneuploid cells in normal tissue and in tumors. Aneuploid cells may arise directly from diploid cells through errors in chromosome segregation, as a consequence of incorrect microtubule-kinetochore attachments, or through failure of the spindle checkpoint. A second route to formation of aneuploid cells is through a tetraploid intermediate, where division of tetraploid cells can yield very high rates of chromosome missegregation as a consequence of multipolar spindle formation. Diploid cells may become tetraploid through a variety of mechanisms, including endoreduplication, cell fusion, and cytokinesis failure. Although aneuploid cells may arise from either diploid or tetraploid cells, the fate of the resulting aneuploid cells may be distinct. It is therefore important to understand the different pathways that can give rise to aneuploid cells, and how the varied origins of these cells affect their subsequent ability to survive or proliferate.     

DNA content analysis of peripheral blood B-lymphocytes in plasma cell malignancies

A double fluorescence assay has been employed for the detection of cell surface and/or cytoplasmic immunoglobulins (Ig) and the measurement of nuclear DNA content in the same cell. Following staining for Ig by means of FITC conjugated antibodies directed against heavy or light chains, cell suspensions or cytospin preparations were ethanol fixed and stained with a propidium iodide-RNAse solution. In this way, the cytometric DNA content of circulating B-lymphocytes was analyzed in three patients suffering from plasma cell malignancies with an excess of peripheral blood B-lymphocytes and evidence of aneuploid bone marrow plasma cells. Aneuploid circulating B-lymphocytes with the same DNA stem-line as bone marrow plasma cells were found in two patients with advanced disease but not in the only one we studied at presentation. Aneuploid lymphocytes had surface immunoglobulins bearing the same light chain as the M-protein. In addition, a significant percentage (23%) of cells lacking either surface or cytoplasmic immunoglobulins proved to be aneuploid in plasma cell leukemia. Nuclear DNA measurement combined with surface or cytoplasmic marker analysis appears to be a reliable method for studying neoplastic lymphoid precursor cells in plasma cell malignancies.     

DNA ploidy and survival in breast cancer patients

Flow cytometric DNA ploidy measurements using frozen or deparaffinized tumor specimens were performed on 565 primary breast cancers from patients treated in the period 1975-1984. Twenty-nine percent of the cases were diploid, 61% had a single aneuploid stemline, and 10% were multiploid. Aneuploid tumors more often had negative estrogen receptor values than diploid tumors, but no significant correlation was found between ploidy class and TNM stage. Patients with more than ten positive axillary lymph nodes had predominantly aneuploid tumors. Overall and distant relapse-free survival were higher for patients with diploid tumors and low-aneuploid tumors. Stratification of the patients according to degree of lymph node involvement, TNM stage, and menopausal stage showed that the prognostic effect of aneuploidy was apparent predominantly in patients with locally advanced disease. Postmenopausal node-positive patients with diploid tumors had a significantly better prognosis than those with aneuploid tumors, but this difference was not found for the comparable premenopausal group. Multivariate analysis with the Cox proportional hazards model indicated that ploidy is an additional, independent prognostic factor in postmenopausal patients.     

Aneuploidy and inbreeding depression in random mating and self-fertilizing autotetraploid populations

Summary Almost all autotetraploids produce aneuploid progeny because of irregularities at meiosis. Aneuploid plants produce high frequencies of aneuploids. If it were not for selection against aneuploid gametes and sporophytes the amount of aneuploidy would increase every generation. Most experimental and theoretical studies on population genetics and heterosis in autotetraploids have neglected aneuploidy as a factor. To take aneuploidy into account experimentally requires the cytological identification of all chromosomes and to consider it theoretically requires a huge amount of computations. Consequently, microcomputer programs have been devised to show the effects of random mating and self-fertilization in autotetraploid populations. According to the model aneuploidy rapidly increases in randomly mated and self-fertilized autotetraploid populations until they achieve an equilibrium where the amount of aneuploidy introduced into the population is balanced by the amount of aneuploidy removed from the population by selection. The model suggests that self-fertilized populations have greater frequencies of aneuploid gametes and zygotes than do randomly mated populations and therefore aneuploidy may be a significant cause of the great inbreeding depressions found in autotetraploids.Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 9998     

Detection of residual aneuploid leukemic cells by "continuous gating".

BACKGROUND: A new method for the detection of residual aneuploid leukemic cells in bone marrow by flow cytometry is described. This method is based on the analysis of FCM derived list-mode-datasets with a new software called "Continuous Gating". The program is able to decrease the detection level of aneuploid tumor cells by analyzing groups of cells with comparable antigen density and scatter properties. METHODS: Aneuploid acute lymphocytic leukemia cells with a known CD34 expression were diluted with diploid bone marrow cells to a concentration of 10, 1, 0.1, 0.05, and 0.01%. Each sample was measured in a FACScan flow cytometer, after staining with CD34 Moab and propidium iodide. Listmode-data were analyzed with the new "Windows"-based "Continuous Gating" software. A gate was set in the DNA parameter, defining the channels in which the aneuploid G0/G1-peak of possible residual tumor-cells should be found. Ten thousand overlapping gates of size 200 x 200 channels (out of 1,023 x 1,023 channels) were set automatically by the program into the side-scatter (SSC)/CD34 dot-plot, calculating the percentage of aneuploid G0/G1-phase cells for every specific gate. RESULTS: The results are plotted in a contour-plot. In dot-plot gates with less than 20 cells, the calculation of the percentage of aneuploid cells was declared invalid and the area in the contour-plot was marked. Detection of residual aneuploid cells, based on a defined expression of CD34 and granularity (SSC), was possible down to a contamination of 0.1%. CONCLUSIONS: The new "Continuous Gating" software can be used for the automated detection of aneuploid leukemic cells, if the density of a certain surface-marker is slightly different from normal cells.     

Chromosomal variations within aneuploid cancer lines.

Aneuploid cancers exhibit a wide spectrum of clinical aggressiveness, possibly because of varying chromosome compositions. To test this, karyotypes from the diploid CCD-34Lu fibroblast and the aneuploid A549 and SUIT-2 cancer lines underwent fluorescence in situ hybridization (FISH) and DAPI counterstaining. The number of DAPI-stained and FISH-identified chromosomes, 1-22, X,Y, as well as structural abnormalities, were counted and compared using the chi(2), Mann-Whitney rank sum test and the Levene's equality of variance. Virtually all of the evaluable diploid CCD-34Lu karyotypes had 46 chromosomes with two normal-appearing homologues. The aneuploid chromosome numbers per karyotype were highly variable, averaging 62 and 72 for the A549 and SUIT-2 lines, respectively. However, the A549 chromosome numbers were more narrowly distributed than the SUIT-2 karyotype chromosome numbers. Furthermore, 25% of the A549 chromosomes had structural abnormalities compared to only 7% of the SUIT-2 chromosomes. The chromosomal compositions of the aneuploid A549 and SUIT-2 cancer lines are widely divergent, suggesting that diverse genetic alterations, rather than chance, may govern the chromosome makeups of aneuploid cancers.     

Flow cytometric analysis of DNA aneuploidy in primary and metastatic human solid tumors

DNA histograms were measured by flow cytometry for 656 human solid tumors (365 primary and 291 metastatic). The proportion of aneuploid cells in cell suspensions obtained by mechanical disaggregation was significantly higher than those obtained after enzymatic disaggregation (collagenase + DNAse) of the same tumor. A strong correlation was observed between the values of DNA-indices measured after staining with propidium iodide and with 4',-6-diamidino-2-phenylindole (r = 0.97). Aneuploid cells were observed in 430 tumors (66%); 30 of these had two aneuploid stemlines, and two had three aneuploid stemlines. The overall frequency of aneuploidy was 61% among primary and 71% among metastatic tumors. The median value of the DNA index was 1.67 for 224 primary aneuploid tumors and 1.68 for 206 metastatic aneuploid tumors. For most diseases, the largest proportion of aneuploid primary and metastatic tumors had DNA-indices in the hypertriploid region. No major differences in frequency and degree of aneuploidy was observed between primary and metastatic tumors. For carcinomas of the bladder and prostate, frequency of aneuploidy was higher among poorly differentiated, than among moderately and well-differentiated tumors. For carcinomas of the breast and for sarcomas, tumors with DNA-indices of greater than 2.0 were observed mostly in the poorly differentiated group. For patients with carcinomas of the bladder and prostate most tumors at earlier stages of disease were diploid; whereas most tumors at later stages of disease were aneuploid. For patients with carcinomas of the ovary, colon, and kidney, no relationship between stage of disease and aneuploidy was evident.     

Gene expression and distribution of Swi6 in partial aneuploids of the fission yeast Schizosaccharomyces pombe

Imbalances of gene expression in aneuploids, which contain an abnormal number of chromosomes, cause a variety of growth and developmental defects. Aneuploid cells of the fission yeast Schizosaccharomyces pombe are inviable, or very unstable, during mitotic growth. However, S. pombe haploid cells bearing minichromosomes derived from the chromosome 3 can grow stably as a partial aneuploid. To address biological consequences of aneuploidy, we examined the gene expression profiles of partial aneuploid strains using DNA microarray analysis. The expression of genes in disomic or trisomic cells was found to increase approximately in proportion to their copy number. We also found that some genes in the monosomic regions of partial aneuploid strains increased their expression level despite there being no change in copy number. This change in gene expression can be attributed to increased expression of the genes in the disomic or trisomic regions. However, even in an aneuploid strain that bears a minichromosome containing no protein coding genes, genes located within about 50 kb of the telomere showed similar increases in expression, indicating that these changes are not a secondary effect of the increased gene dosage. Examining the distribution of the heterochromoatin protein Swi6 using DNA microarray analysis, we found that binding of Swi6 within ~50 kb from the telomere occurred less in partial aneuploid strains compared to euploid strains. These results suggest that additional chromosomes in aneuploids could lead to imbalances in gene expression through changes in distribution of heterochromatin as well as in gene dosage.     

两广茶区10个茶树品种染色体数目研究

采用植物染色体去壁、低渗、火焰干燥制片技术,分析了两广茶区１０个茶树品种染色体数目。结果表明,这１０个茶树品种均为二倍体（２ｎ＝２ｘ＝３０）;但在广东平远锅　品种中发现３个三倍体细胞;在广东连南大叶种中发现１个单倍体细胞;在清远笔架茶等６个茶树品种中均发现染色体数少于３０条的细胞。研究结果说明,茶树染色体基数具有高度的稳定性,极端气候条件的变化,很可能是导致染色体出现整倍性或非整倍性变异的原因。