MLPA-微阵列技术在Y染色体异常检测中的初步应用

摘要: 为了探讨MLPA-微阵列技术用于检测性染色体异常的可行性和精确性, 针对Y染色体上的3个基因TSPY(p11.2)、PRY(q11)和RBMY(q11.2)设计MLPA探针, 应用MLPA-微阵列技术对15例已知染色体核型的样品进行检测, 将检测结果与各样品核型分析和PCR的检测结果进行对照和比较。结果表明, MLPA-微阵列技术对上述各基因位点的检测结果与样品染色体核型基本吻合, 特别是对二例核型分析没有获得染色体结构异常信息的样品, MLPA-微阵列技术检测出Y染色体微小的缺失或指示某些未知染色体片段的信息, 并与PCR检测结果完全相符。表明文章报道的MLPA-微阵列技术能够检测核型分析无法分辨的微小变化和异常, 显示MLPA-微阵列技术在染色体异常分析中具有很高的检测效率和准确性, 相对于染色体核型分析具有明显的优势, 在临床染色体病诊断中具有较大的应用前景。     

水稻染色体的长度顺序和编号问题

水稻染色体形体较小,很难从形态上区分各别染色体。尽管有许多学者报道了水稻核型的研究结果,但不同学者之间的分歧依然存在。本文对不同学者所作核型进行了分析,从而对现行的水稻染色体编号系统提出了新的看法。     

水稻(Oryza sativa)核型分析

水稻(Oryza sativa)核型分析结果:在12对染色体中,具中部着丝点的有5对,近中部着丝点的有6对(包括随体染色体),1对近端部着丝点。本文还着重讨论了随体的数目及所在的染色体。     

水稻(Oryza sativa)核型分析

水稻(Oryza sativa)核型分析结果:在12对染色体中,具中部着丝点的有5对,近中部着丝点的有6对(包括随体染色体),1对近端部着丝点。本文还着重讨论了随体的数目及所在的染色体。     

在水稻体细胞愈伤组织中进行核型的分析

国内外不少学者对水稻(O. sativa)2n=24染色体的形态进平行了研究,大多数是采用种子萌发后的根尖作为适宜材料。由于水稻染色体较小,核型分析需要一定量的种子,而珍贵的远缘杂种(如栽培稻与野生稻杂交后代)结实率极低,所结种子极少。为了克     

雄蚕终变期染色体核型分析

关于家蚕 Bombyx mori L.染色体核型分析,以粗线期染色体(蒋同庆和邓一民,1985;Rasmussen,1976;Traut,1976)及联会复合体(马昆和施立明,1988;王宗舜等,1981)研究较多,中期细胞染色体仅见按径率进行的核型分析(李振刚等,1980),迄今未见有关终变期染色体研究的报道。本实验发现终变期染色体具有显著形态特征,适于核型分析,并对该期的染色体作了初步分析。     

纯合等臂假双着丝粒X染色体致性腺发育不良1例病例报告与文献复习

目的提高对纯合等臂假双着丝粒X染色体特纳综合征的鉴别诊断和临床特征的认识。方法用染色体核型分析技术,对1例14岁女性发育迟缓、先天子宫及双侧卵巢缺如患者的外周血细胞做细胞遗传学检查;用荧光原位杂交(FISH)和染色体微阵列芯片技术(chromosome microarray assay, CMA)进行验证。结果:发现该患者染色体核型为纯合等臂假双着丝粒X染色体;FISH检测在间期细胞中确认有3个X染色体着丝粒,在中期细胞中确认了其中2个着丝粒位于同一条染色体;CMA检测确认了Xpter-Xq22.1的片段重复及Xq22.1-Xqter的片段缺失。结论确诊患者为特纳综合征;纯合等臂双着丝粒X染色体的TS患者比较罕见,染色体核型分析、FISH及CMA技术的合理组合应用,有助于鉴别诊断并可能指导早期治疗。     

大绒鼠的分带核型研究

本文采用G带、C带和银染核仁组织者(Ag-NORs)等技术,对大绒鼠(Eothe nomys miletus miletus)的核型进行观察分析。结果表明:2n=56,常染色体和性染色体皆为单臂染色体。X染色体的长度接近于No.1染色体,Y染色体的长度相当于14号染色体。G分带可鉴别每对染色体的特征,C-带核型中全部着丝点C带均显示不同程度的阳性。Y染色体整条呈阳性。Ag-NORs有5对,分别分布于1、2、6、14和27号染色体的着丝粒附近。通过核型分析,对大绒鼠的分类地位进行了初步探讨。     

不同基因型割手密无性系的核型分析

为探讨不同基因型割手密无性系间的亲缘关系,采用酶解去壁低渗法对不同基因型的割手密材料进行核型分析。10份供试材料的绝大多数染色体为中部着丝点(m)染色体,少数为近中部着丝点(sm)染色体及正中部着丝点(M)染色体,部分材料中还具有端部着丝点(T)染色体及近端部着丝点区染色体(t);依据分析结果总结了参试材料的核型公式;参试材料中有2份为1B核型、7份为2B核型、1份为2C核型。参试材料间的核型均存在差异且不对称。     

黑龙江地区466例孕妇羊水穿刺产前诊断结果分析

目的:探讨羊水细胞染色体异常核型与各产前诊断之间的关系。方法:466例高危孕妇行羊膜腔穿刺术后羊水细胞培养及染色体核型分析。结果:异常核型66例,异常率14.16%,包括染色体数目异常27例,三体综合征22例(21-三体15例、18-三体6例、13-三体1例),占异常染色体核型的33.33%,占染色体数目异常的81.48%;染色体结构异常39例,主要包括染色体多态性、平衡易位、倒位和衍生等,占染色体异常核型的59.10%。异常核型检出率中血清学筛查高危组(14.44%)要高于高龄妊娠组(10.89%)和有不良孕产史组(11.11%)(P0.05);超声提示胎儿发育异常组(23.26%)要高于血清筛查高危组(P0.05)。结论:血清筛查高危和超声提示胎儿发育异常是黑龙江地区最主要的产前诊断指征,异常核型以21-三体综合征检出率最高。通过对高危孕妇羊水细胞染色体的核型分析可发现部分染色体疾病,从而避免此类出生缺陷儿的出生。     

BrdU-Hoechst-Giemsa方法的进一步改进以及青鱼和草鱼复制带核型的研究

对BrdU-Hoechst-Giemsa方法进行了一定的改进和补充;并将它应用于青鱼和草鱼的核型研究中。实验进一步阐明,BrdU-Hoechst-Giemsa方法的关键性步骤之一是要先测算实验鱼的细胞周期;BrdU、AMD、Hoechst 33258,EB和AO虽都有抑制鱼类染色体浓缩、促进染色体分带的作用,但其中以DNA碱基特异性给合物BrdU、AMD和Hoechst 33258较佳,特别用AMD和Hoechst 33258同时处理活细胞的分带效果最好。     

Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high-resolution chromosome banding

Ethidium bromide (EB) is known to intercalate between stacked base pairs without specific base-pair preference. Its use in cultured human lymphocytes and Burkitt's lymphoma cells resulted in the accumulation of cells in prophase and prometaphase stages. Inhibition of mitotic chromosome condensation as a possible mechanism involved in this phenomenon is discussed. A simple method for obtaining high-resolution banding patterns on elongated chromosomes was devised as follows: Human lymphocytes cultured for 3 days with phytohemagglutinin were exposed to EB (5-10 micrograms/ml) and Colcemid (0.02 micrograms/ml) simultaneously for 2 h and then routinely harvested for chromosome preparation. High-resolution G-bands were obtained by Giemsa staining following mild trypsin treatment.     

Characterization of human MAPRE genes and their proteins

The MAPRE genes encode the EB1 family proteins. The yeast EB1 protein had been shown to play important roles in microtubule dynamic regulation, cytokinesis, mitotic spindle positioning, and episome segregation. To facilitate functional studies of mammalian EB1 family proteins, we characterized the human MAPRE genes (MAPRE1, MAPRE2, and MAPRE3) and their proteins (EB1, RP1, and EBF3). We found that the three MAPRE genes had similar genomic structures but were on different chromosomes. We showed that EB1 family proteins appeared to be expressed ubiquitously. We identified two EBF3 proteins, which were encoded by alternatively spliced MAPRE3 mRNAs. We demonstrated that there were also two RP1 proteins, which were products of translation from different initiation codons. We showed that the three EB1 family proteins had different abilities to interact with APC in vitro, and we provided the first direct evidence for the association between endogenous EB1 and APC.     

Karyotype characteristics of C3H10T1/2-strain mouse fibroblasts undergoing long-term selection for their capacity to multiply in the presence of ethidium bromide

Variants Br-0.5 and Br-1 of minimally transformed mouse fibroblasts of C3H10T1/2 line were selected for their ability to proliferate in the medium with 0.5 and 1 mkg/ml of ethidium bromide (EB) toxic for cells of the parent line. Karyological analysis of metaphase chromosomes, stained by Giemsa for G-bands, revealed the number of significant changes in the karyotype of cells resistant to EB. In cells of the resistant sublines the variability of chromosomes was higher than in those of the sensitive population. Two groups of cells are distinguished in the Br-0.5 subline: those with near-diploid and tetraploid chromosome numbers, respectively. The number of polyploid cells in the EB-resistant sublines increases up to 38%, compared to 2% in the parent population. The marker chromosomes in resistant cells originated from translocations, deletions and inversions, with preferential involvement of the material from chromosomes 1.4 and 6. The pericentromeric region of chromosome 4 and the distal region of chromosome I (region 1H1-1H6) were characterized by the increased variability and preferential involvement in rearrangements. In cells of both resistant sublines double mini-chromosomes (1-5 copies per cell) were found. The relation between the revealed chromosomal rearrangements and the mechanism of EB-resistance is discussed.     

High resolution mid-prophase human chromosomes induced by echinomycin and ethidium bromide

The antitumor antibiotic echinomycin (EM) was used in combination with ethidium bromide (EB) to induce high resolution mid-prophase chromosomes from amethopterin synchronized blood lymphocytes. With this combination of drugs, trypsin G-banded chromosomes showed over 1200 bands per haploid set in cells having a mid-prophase length of decondensation.     

Cloning and sequence analysis of an Ig lambda light chain mRNA expressed in the Burkitt''s lymphoma cell line EB4.

A cDNA library was constructed from the mRNA of the Ig lambda producing Burkitt's lymphoma cell line, EB4. Overlapping clones encompassing the coding sequence of the Ig lambda mRNA were isolated and sequenced. The predicted amino acid sequence shows a short hydrophobic leader peptide and a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The V region belongs to subgroup VI and has greatest homology (80%) with the Amyloid-AR protein. The constant region is the Kern- Oz+ isotype. Probing normal human DNA with the subcloned V lambda coding sequence detects one gene at high stringency and a family of 11 members at low stringency. To date, no restriction enzyme site polymorphisms have been detected. The V lambda VI gene is rearranged on both chromosomes of EB4 and is deleted on both chromosomes in the Burkitt's lymphoma cell line BL2.     

Whole‐genome association study for energy balance and fat/protein ratio in German Holstein bull dams

The metabolic status of cows is important to health and fertility, especially in early lactation, and energy balance (EB) and fat/protein ratio (FPR) are considered as appropriate indicators for metabolic disorders. The aim of this study was to detect SNPs (single nucleotide polymorphisms) associated with EB and FPR in German Holstein bull dams belonging to the research herd Karkendamm. Bull dams were genotyped using the Illumina Bovine SNP 50K Bead chip^® comprising 54 001 SNPs. A total of 43 593 SNPs and 586 (EB) and 668 (FPR) bull dams passed the quality control criteria. Phenotypes were deregressed breeding values estimated via random regression animal models for lactation days 11, 20, 30, and 42 for EB and FPR. Whole‐genome association analyses were carried out fitting principal components as covariates to adjust for genetic substructure. Permutation tests were applied to estimate genome‐wise significance. Across all observed lactation days, 19 SNPs located in four different intervals on chromosomes 1, 14, 16, and 27 were detected. For EB, seven markers across four chromosomes were identified. There was no overlap between markers associated with FPR and EB. SNPs associated with FPR were mostly located in QTL regions for milk production traits, especially in the region of DGAT1, whereas SNPs associated with EB mainly showed positional relationships to previously described QTL regions affecting functional traits, especially fertility.     

应用荧光原位杂交检测EB病毒BNLF-1基因在转基因小鼠子代染色体上的整合及其定位

应用荧光原位杂交技术研究了EB病毒潜伏膜蛋白基因(BNLF-1)在转基因小鼠子二代染色体上的整合及其定位。结果在两只子二代转基因小鼠中,分别观察80个和60个分裂相,出现杂交信号的核型分别为27和18个,检出率为33.8％和30％。转基因分别整合在14号染色体和10号染色体上。提示转基因BNLF-1已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给子代;推测转基因原代鼠的转基因整合可能是随机的多位点整合。     

Critical role for the EB1 and APC interaction in the regulation of microtubule polymerization

Human EB1 was originally cloned as a protein that interacts with the COOH terminus of adenomatous polyposis coli (APC). Interestingly, this interaction is often disrupted in colon cancer, due to mutations in APC. EB1 also interacts with the plus-ends of microtubules and targets APC to microtubule tips. Since APC is detected on the kinetochores of chromosomes, it has been hypothesized that the EB1-APC interaction connects microtubule spindles to the kinetochores and regulates microtubule stability. In yeast, EB1 regulates microtubule dynamics, and its binding domain in APC may be conserved in Kar9, an EB1 binding protein involved in the microtubule-capturing mechanism. These results suggest that the interaction of EB1 and APC is important and may be conserved. However, it is largely unknown whether the EB1-APC interaction affects microtubule dynamics. Here, we show that EB1 potently promotes microtubule polymerization in vitro and in permeabilized cells, but, surprisingly, only in the presence of the COOH-terminal EB1 binding domain of APC (C-APC). Significantly, this C-APC activity is abolished by phosphorylation, which also disrupts its ability to bind to EB1. Furthermore, yeast EB1 protein effectively substitutes for the human protein but also requires C-APC in promoting microtubule polymerization. Finally, C-APC is able to promote microtubule polymerization when stably expressed in APC mutant cells, demonstrating the ability of C-APC to promote microtubule assembly in vivo. Thus, the interaction between EB1 and APC plays an essential role in the regulation of microtubule polymerization, and a similar mechanism may be conserved in yeast.     

Microfluorimetric analysis of dynamics of genomic changes of Chinese hamster fibroblasts CHL V-79 RJK with multidrug resistance

Results of karyological analysis of cells CHL V-79 RJK selected for resistance to ethidium bromide (EB) causing multidrug resistance (MDR) (line Vebr-5) were compared with the data of microfluorimetric determination of DNA content in individual chromosomes of the karyotype. The analysis was performed at the 11th and 88th passages. Karyotyping of Vebr-5 has shown the presence of an additional genetic material (ADM) in the form of homogenously or differentially stained regions (HSRs and DSRs, respectively) in two chromosomes (Z1 and Z6, loci 1 p29-31 and 1q26, respectively). HSRs in Z6, in the region of localization of the wild type of gene mdr, had unstable length and structure characteristic of morphological markers of amplification of genes of the family mdr. During long cultivation of Vebr-5 in the presence of EB (88 passages), the instability of HSRs in Z6 increased. Results of microfluorimetric analysis of Vebr-5 at the 11th passage have shown an increase in the DNA content not only in chromosomes Z1 and Z6 marked by HSRs, but also in three chromosomes (Z5, Z12 and Z13) that have no visual morphological changes. The corresponding analysis at the 88th passage has also revealed non-random changes in the DNA content in four more chromosomes: an increase in Z14, while a decrease in chromosomes 8, Z7, and Z9. A decrease of the DNA content in chromosomes is considered to be a result of a partial loss of genetic material, while its increase is a result of its translocation and (or) amplification. Coefficient of variation of the DNA content changes for large chromosomes amounted to about 9%. while for small chromosomes it is about 26%, which indicates that small chromosomes have greater potential for instability than the large ones. The data obtained not only confirm, but also enlarge the concept of directions and character of destabilization of the cell genetic apparatus in the process of neoplastic transformation due to the MDR acquisition by cells.