基因组印迹的起动与沉默

在配子中基因组印迹起动复合物的结合引发印迹现象,印迹起动复合物中有多种可鉴定成分,存在结合的精确时间和机制,印迹起动复合物似乎仅出现在生殖细胞系,而甲基－CpG－结合蛋白则能延续增殖印迹。因此基因组印迹调节着发育基因或组织特异性单等位基因的瞬时表达及大染色体结构域中基因之间的相互作用。     

脂质体介导含IL-2及IFN-γ cDNA腺相关病毒质粒的转基因及抗肿瘤作用

h IL- 2基因和 m IFN- γ基因经 IRES连接后克隆入腺相关病毒质粒表达载体 p AC中 ,构建得双基因质粒表达载体 p AC- FRI.体外经阳离子脂质体 Dosper介导转染小鼠肝癌细胞 MM45T.Li,Northern印迹及生物活性检测分别从 RNA水平和蛋白质水平证明了 2个基因的表达 .直接瘤内注射 Dosper- DNA复合物后 ,与对照组 ( Lac Z)相比 ,双基因组及 IL- 2或 IFN-γ单基因组均产生了较明显的抗瘤作用 ,并诱发了较高的特异 CTL活性 .     

基因组印迹与种子发育

胚乳介导营养物质从母体到胚的转运过程,是开花植物中发生印迹的重要部位。胚乳的发育异常会导致胚的败育。在拟南芥中已鉴定到三个FIS (fertilization-independent seed) 基因,能制止无需受精即形成种子的发育过程,即FIS1/ MEDEA、FIS2和FIS3/FIE。其中MEDEA基因是胚乳发育的主要调控基因,在胚乳中被印迹。FWA基因也在胚乳中被印迹。系统阐述了植物基因组印迹的机理以及MEA和FWA印迹机制的研究进展,并介绍了印迹发生的亲本冲突学说、印迹的方式及其它已报道的印迹基因。     

真核转录因子TFⅡD研究进展

结合转录因子 TFⅡD 结构与功能,论述了 RNA 聚合酶Ⅱ是如何从一个特异基因的转录起始点起动转录的.转录因子 TFⅡD 是一种序列特异的 DNA 结合蛋白因子,它首先与含 TATA 的启动子形成前起始复合物,后者指导 RNA 聚合酶Ⅱ和其它基本转录因子最终组装成转录起始复合物.很多转录激活因子均通过与 TFⅡD 相互作用,控制转录起始复合物的组装或影响其稳定性,调节基因转录.因此,TFⅡD是一种极其重要的基本转录因子.     

以酵母单杂交体系克隆水稻RAPB基因cDNA及其序列测定

在一些真核基因的 5′上游区中存在核心序列为CCAAT的顺式元件 ,CCAAT结合蛋白以异源多聚体的方式结合于该顺式元件并行使转录调控功能 .CCAAT结合复合物至少存在 3个不同的亚基 ,且结合复合物的单个亚基都不具备DNA结合活性 .首次报道以酵母单杂交体系筛选方法 ,结合酵母功能互补法鉴定 ,从水稻中克隆了定名为RAPB的cDNA ,它编码与酵母CCAAT结合复合物中HAP2亚基具类似功能的蛋白 .RAPB蛋白的C端同样存在与HAP2功能域高度保守的区域 ,但其N端与其他HAP2类似蛋白间无明显的顺序同源性 ,且不存在谷氨酰胺丰富区 .根据Southern杂交结果推测 ,在水稻 (OrizasativaL .)基因组中仅存在一个拷贝的RAPB基因 .     

同源异型盒基因Emx在人胎盘绒毛膜中的扩增

以鹌鹑Emx cDNA片段作为探针,对人胎盘绒毛膜细胞和成人血细胞的基因组DNA进行DNA印迹分析. 结果表明,在人胎盘绒毛膜细胞中Emx基因剂量较成人血细胞高6倍,显示Emx基因在人胎盘绒毛膜细胞基因组中发生了扩增.     

腺相关病毒介导基因组来源的基因转移与整合

构建一个带β-珠蛋白基因组序列的腺相关病毒载体AV53HS2Δβ2Neo.经包装成重组腺相关病毒后,转导红系细胞.DNA印迹证实包含红系增强子、β-珠蛋白基因和筛选标志基因的前病毒基因组完整整合于红系细胞基因组中.结果说明腺相关病毒载体能介导基因组序列来源的目的基因稳定整合于受体细胞基因组中.     

抗菌肽B基因导入水稻及转基因植株的鉴定

构建了一个适合在水稻中表达的含有抗菌肽B基因的转化载体(pCB1),应用基因枪转化法将其导入水稻未成熟胚,获得了一些转基因水和植株.Basta抗性鉴定,抗菌肽B基因PCR扩增分析,点渍印迹和Southern印迹分析结果表明,选择标记基因(bar)和抗菌肽B基因都已整合入转化水稻基因组中,Northern印迹分析证实了抗菌肽B基因在RNA水平上的表达.转基因水稻植株增强了对水稻白叶枯病和细条病的抗性.     

印记基因DLK1的研究进展

在哺乳动物中,有一部分特别的基因,它们由于受到印迹而只表达单一亲本的基因,这种表观遗传的修饰现象就是基因组印记,这有别于经典的孟德尔遗传学定律。DNA甲基化是一种重要的表观遗传修饰,主要的修饰部位发生在DNA的CpG岛。它参与了细胞分化,基因组稳定性、基因印记等多种细胞生物学过程,基因印迹的建立和维持是胚胎正常发育的基础,这一过程的实现有赖于各种DNA甲基化转移酶的精确表达和密切的配合。已发现在哺乳动物的基因组中存在着许多的印记基因,DLK1基因为父系表达母源沉默的印记基因,它的表达同样受到DNA甲基化的调节,它首先在神经母细胞瘤发现并克隆,定位于人类染色体14q32,属于表皮生长因子样超家族的成员之一,约有6个外显子。研究表明,DLK1基因在胚胎肝、早期肌肉组织以及造血干细胞等组织中均有表达,人DLK1基因全长1557bp,编码序列含有1152核苷酸,编码383个氨基酸残基,在人、小鼠、绵羊都存在保守序列,它参与多种细胞的增殖、分化并且与相关肿瘤的发生发展有着密切的关系,印迹基因的印迹异常与肿瘤的易感性及发生发展有重要的关系,本文就国内外DLK1基因的研究进展做一综述。     

c-flag-ago3质粒在人293细胞系中的表达及AGO3蛋白复合物的细胞定位

将c-flag-ago3质粒转入人293细胞系中,使c-flag-ago3基因稳定表达,为进一步研究AGO3蛋白复合物的结构、功能奠定了基础.利用亲和标签flag对目标蛋白进行检测和监测,将已合成的c-flag-ago3质粒和用于对照的质粒si-ago3(能使ago3基因沉默的质粒)导入人293细胞系中,在荧光镜下观察转染效果.RT-PCR法检测基因含量,蛋白印迹法(WB)检测人293细胞表达的flag-ago3,并对其蛋白复合物进行细胞定位.结果显示,c-flag-ago3质粒和si-ago3质粒成功地导入人293细胞系中,免疫细胞化学显示c-flag-ago3基因在细胞中高表达、并检测到AGO3复合物定位于细胞质中.AGO3复合物在细胞中可稳定表达,为进一步研究AGO3蛋白复合物的构成及其在人体中的功能奠定了基础.     

The uniqueness of the imprinting mechanism

An epigenetic imprinting mechanism that is based on a gamete-specific methylation imprint restricts expression of a subset of mammalian genes to one parental chromosome. Recent results suggest that imprints may act only indirectly to induce monoallelic expression of coding genes. Instead, atypical non-coding RNAs appear to be a primary target of the imprints, and their parental-specific repression correlates with parental-specific expression of linked coding genes.     

Imprint cytology in the diagnosis of Helicobacter pylori. Does imprinting damage the biopsy specimen?

OBJECTIVE: To investigate the efficacy of imprint cytology in the diagnosis of Helicobacter pylori infection and whether it damages the biopsy specimen for subsequent histologic examination. STUDY DESIGN: Two antral biopsies were taken from 76 patients with dyspeptic symptoms undergoing upper gastrointestinal endoscopy. Imprint cytology was made from the first specimen. This specimen was fixed in 10% formalin and sent for histopathologic examination. The second specimen was directly fixed in 10% formalin for routine histopathologic examination without being used for an imprint. The imprint smears were examined by cytopathologists. The biopsy specimens were examined by pathologists who did not know which specimens were used for the imprints. RESULTS: H pylori was seen in smears from 55 (72%) patients and in both biopsy specimens from the same patients. The pathologists could not recognize the biopsy specimens from which the imprints were made. Concordance between imprint cytology and histopathology was 100%. CONCLUSION: Imprint cytology is a suitable test for H pylori diagnosis, and imprints do not adversely affect the quality of the biopsy specimen.     

Genomic imprinting in Drosophila is maintained by the products of Suppressor of variegation and trithorax group,but not Polycomb group,genes

Genomic imprinting is a form of epigenetic inheritance that is characterized by differential expression of a gene depending on its parental origin. The mini-X chromosome Dp(1;f)LJ9 in Drosophila shows this type of classical imprinting; when transmitted by the maternal parent genes on this chromosome are fully expressed, but when the chromosome is transmitted by the male parent at least three genes are subject to silencing, resulting in a variegated expression pattern. Chemical and environmental modifiers of position-effect variegation have been shown to alter the somatic maintenance of the imprint. To extend these observations, several mutations in chromatin-associated proteins were examined for their effect on imprinting on the Dp(1;f)LJ9 mini-X chromosome. Effects on establishment and maintenance were independently assessed by genetically associating the mutations in chromatin modifiers with the mini-X chromosome in either the parents, where the imprint is established, or the progeny, in which the imprint must be maintained. Nine Suppressor of variegation [ Su(var)] mutations, including alleles of the Su(var)2-5 gene, which encodes the well characterized heterochromatin-associated protein HP1, abolished maintenance but not the establishment of the imprint. Mutant alleles of two genes in the trithorax group ( trx-G), brahma and trithorax, showed a maternal-effect enhancement of the paternal imprint. Surprisingly, however, with the exception of an Enhancer of Polycomb [ E(Pc)] allele, none of the Polycomb-group ( Pc-G) mutations tested affected the imprint. Thus, the maintenance of this imprint relies on the wild-type products of Su(var) and trx-G, but not Pc-G, genes. Finally, none of the mutations tested affected the maintenance of the maternal imprint or the establishment of either the maternal or paternal imprint, suggesting that the maternal and paternal imprints depend on different molecular processes and that imprint establishment and maintenance are independently regulated.     

Utility of imprint cytology for early presumptive diagnosis in clinically suspicious cervical cancer

OBJECTIVE: To determine the utility of imprint cytology (IC) in providing an early presumptive diagnosis of clinically suspected cervical carcinoma. STUDY DESIGN: A total of 219 clinically suspicious cervical cancer cases underwent Pap test, punch biopsy and IC at the same sitting. Correlations were performed between these diagnostic modalities to determine the sensitivity and specificity of IC in diagnosis of cervical cancer. RESULTS: The overall accuracy of IC in detecting cervical cancers was 96.2%. About 78% of squamous cell carcinomas (SCC), 60% of adenocarcinomas and 100% of small cell carcinoma could be accurately typed on imprints. Twelve malignant lesions were diagnosed on IC among 26 unsatisfactory biopsies. Although there was no false positive result, 3.5% false negative diagnoses were given on IC. The sensitivity and specificity of imprint smear cytology to detect malignancy was 96.2% and 100%. Agreement between imprint cytology and Pap smear diagnosis of malignancy was 95.3%. kappa Statistics revealed excellent agreement between imprints and biopsies and between imprints and Pap smears in diagnosis of malignant lesions. CONCLUSION: IC can be used as an adjunctive technique for an early and reliable preliminary presumptive diagnosis of cancer of the uterine cervix.     

Imprint cytology of sentinel lymph nodes in breast cancer. Experience with rapid, intraoperative diagnosis and primary screening by cytotechnologists

OBJECTIVE: To evaluate the intraoperative imprint diagnoses of smears from sentinel lymph nodes that had been primary screened by cytotechnologists and to assess the most important causes of false negative (FN) imprint diagnoses. STUDY DESIGN: Material consisted of 429 imprints from sentinel lymph nodes in 211 breast cancer patients that were sent for frozen section examination over 13 months. RESULTS: The mean number of imprints/lymph nodes per patient was 2.02. The mean screening time per imprint was 3.6 minutes. Sixty-six sentinel nodes (16%) from 51 women (24%) were metastatic. Imprints and/or frozen sections were positive in 54 nodes (82%). Imprints were positive in 38 nodes, representing 70% of intraoperative positive nodes and 58% of the total number of positive nodes. Twenty-six of 28 (93%) FN imprints were due to suboptimal sampling. Four of 9 FN macrometastases did not contain diagnostic or suspicious cells/cell groups even on rescreening, whereas a few, and then only 1 diagnostic group were identified in 2/9. There were no false positives. CONCLUSION: Primary screening by experienced cytotechnologists is both rapid and reliable and enabled the diagnosing pathologist to concentrate on the frozen section. The major cause of false negative imprints is sampling, even in macrometastases.     

A KRAB domain zinc finger protein in imprinting and disease

The monoallelic expression of imprinted genes is regulated by DNA methylation marks that originate from the oocyte or sperm. Li et al. (2008) show in this issue of Developmental Cell that the KRAB zinc finger protein Zfp57 contributes to the embryonic maintenance of these imprints. At one locus, Zfp57 is also involved in imprint establishment. These findings provide a mechanistic interpretation for Mackay et al.'s recently reported ZFP57 mutations in patients with transient neonatal diabetes.     

The value of lymph node imprint cytodiagnosis: an assessment of interobserver agreement and diagnostic accuracy

The value of lymph node imprint cytodiagnosis: an assessment of interobserver agreement and diagnostic accuracy
The aim of this study was to assess the reliability of cytodiagnosis of lymph node imprints without fixed tissue sections. One hundred randomly selected archival cases were used in the study. These air‐dried May–Grünwald–Giemsa imprint slides were assessed independently and blind by three pathologists. Cases were assigned to one of four diagnostic categories: reactive changes, non‐Hodgkin's lymphoma (NHL), Hodgkin's disease (HD) and secondary malignancy. Each broad diagnosis was compared with the 'correct' reviewed histological diagnosis to calculate interobserver agreement and diagnostic accuracy. The overall κ score (+0.59) was indicative of moderate agreement. The mean pathologist diagnostic accuracy was 78%, with complete agreement with the histological diagnosis in 61% of cases. The main diagnostic difficulties were in the distinction between reactive changes and NHL and distinguishing NHL from HD. Further diagnostic classification, e.g. typing of lymphomas and subclassification of Hodgkin's disease, was not found to be reliable using the imprints alone. With these limitations in mind, pathologists should be able to use lymph node imprints for cytodiagnosis in selected cases. The study also emphasized the utility of imprints as a corollary to the histology and as a tool for cytology training and continuing education.