斑马鱼中囊胚过渡调控机制

斑马鱼中囊胚过渡(MBT)始于受精卵的第10次卵裂,此时亦伴有细胞周期延长,分裂同步性丧失,合子型基因开始转录活化,胚胎细胞开始具备运动迁移能力等现象。斑马鱼MBT。的发生依赖于胚胎细胞的核质比,胚胎细胞周期中的G1时相则只有在合子型基因组开始被转录活化后才能出现。细胞周期检验点的激活可能也是受转录调控的,但中期检验点对DNA复制抑制状态的响应不仅在MBT前后、甚至在MBT前的不同阶段也可能有具体作用途径的差异。活化的P38蛋白在胚胎中的不对称分布是维持卵裂阶段细胞分裂同步性的关键因素。尽管大规模的合子型基因的表达发生在MBT开始后,也有少数与胚层分化有关的合子型基因是在MBT。前表达的,还有一些既有母型表达也有合子型表达的基因在MBT前后分别参与不同的信号途径来调控胚胎的发育与分化。     

Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes

Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.     

A novel Drosophila, mef2-regulated muscle gene isolated in a subtractive hybridization-based molecular screen using small amounts of zygotic mutant RNA

It is unknown how the general patterning mechanisms that subdivide the mesoderm initiate different pathways of cell differentiation. One route to understanding these events is to isolate and analyse genes specifically expressed early in this differentiation process. I have therefore undertaken a novel molecular screen in Drosophila in a systematic search for such genes. The approach utilised subtractive hybridisation coupled to directional cDNA library construction. Libraries were made from as little as 20 microg total RNA isolated from hand-picked embryos of defined stage of development and genotype. In a one-step procedure, the subtraction was 6.5- to 7.25-h wild-type embryos minus 6.5- to 7.25-h twist (twi) zygotic mutant embryos. A two-step procedure in which maternally expressed sequences were subtracted from each of these cDNA libraries, before subtracting twi from wild-type, increased the subtraction efficiency. It resulted in a cDNA population enriched more than 100-fold for mesodermal cDNAs. This was screened by determination of the embryonic expression pattern of each clone in a high throughput procedure and then DNA sequencing. The method, which is comprehensive and does not discriminate against rarer cDNAs, is generally applicable and calculations show that it would work for just 10 embryos. Analysis of one clone, Dmeso18E, that encodes a putative nuclear protein and fulfils the screen's aims is described. It is novel and its expression is mesoderm-specific, twi-dependent, and early during somatic, visceral, and heart muscle differentiation. Two pivotal regulators of mesoderm development and gene expression are Dmef2 and tinman (tin). Analysis of Dmeso18E expression revealed new aspects to their roles: there are effects of Dmef2 on developing muscle much earlier than hitherto believed, and there is tin-independent gene expression in, and invagination of, prospective midgut visceral muscle cells. Dmeso18E expression is regulated by Dmef2, although some expression is Dmef2-independent. The tin-independent and Dmef2-independent expression of Dmeso18E indicates that it either occupies a link between twi and genes like tin and Dmef2 or it lies in a parallel pathway of gene activation.     

The Drosophila ATM homologue Mei-41 has an essential checkpoint function at the midblastula transition

BACKGROUND: Drosophila embryogenesis is initiated by 13 rapid syncytial mitotic divisions that do not require zygotic gene activity. This maternally directed cleavage phase of development terminates at the midblastula transition (MBT), at which point the cell cycle slows dramatically, membranes surround the cortical nuclei to form a cellular blastoderm, and zygotic gene expression is first required. RESULTS: We show that embryos lacking Mei-41, a Drosophila homologue of the ATM tumor suppressor, proceed through unusually short syncytial mitoses, fail to terminate syncytial division following mitosis 13, and degenerate without forming cells. A similar cleavage-stage arrest is produced by mutations in grapes, which encodes a homologue of the Checkpoint-1 kinase. We present biochemical, cytological and genetic data indicating that Mei-41 and Grapes are components of a conserved DNA-replication/damage checkpoint pathway that triggers inhibitory phosphorylation of the Cdc2 kinase and mediates resistance to replication inhibitors and DNA-damaging agents. This pathway is nonessential during postembryonic development, but it is required to terminate the cleavage stage at the MBT. Cyclins are required for Cdc2 kinase activity, and mutations in cyclin A and cyclin B bypass the requirement for mei-41 at the MBT. These mutations do not restore wild-type syncytial cell-cycle timing or the embryonic replication checkpoint, however, suggesting that Mei-41-mediated inhibition of Cdc2 has an additional essential function at the MBT. CONCLUSIONS: The Drosophila DNA-replication/damage checkpoint pathway can be activated by externally triggered DNA damage or replication defects throughout the life cycle, and under laboratory conditions this inducible function is nonessential. During early embryogenesis, however, this pathway is activated by developmental cues and is required for the transition from maternal to zygotic control of development at the MBT.     

The Caenorhabditis elegans genes egl-27 and egr-1 are similar to MTA1, a member of a chromatin regulatory complex, and are redundantly required for embryonic patterning

We show here that two functionally redundant Caenorhabditis elegans genes, egl-27 and egr-1, have a fundamental role in embryonic patterning. When both are inactivated, cells in essentially all regions of the embryo fail to be properly organised. Tissue determination and differentiation are unaffected and many zygotic patterning genes are expressed normally, including HOX genes. However, hlh-8, a target of the HOX gene mab-5, is not expressed. egl-27 and egr-1 are members of a gene family that includes MTA1, a human gene with elevated expression in metastatic carcinomas. MTA1 is a component of a protein complex with histone deacetylase and nucleosome remodelling activities. We propose that EGL-27 and EGR-1 function as part of a chromatin regulatory complex required for the function of regional patterning genes.     

鼻咽癌上皮细胞株HNE1差异表达基因的分离与鉴定

为了分离鼻咽癌差异表达基因 ,应用抑制性扣除杂交技术 ,在正向抑制性扣除杂交中 ,以鼻咽癌上皮细胞株HNE1cDNA作为检测子 ,以人胚鼻咽上皮细胞cDNA作为驱赶子 ;在反向抑制性扣除杂交中 ,以人胚鼻咽上皮细胞cDNA作为检测子 ,以鼻咽癌上皮细胞株HNE1cDNA作为驱赶子 ,分别通过抑制性扣除杂交 ,构建了鼻咽癌上皮细胞株HNE1表达下调和表达上调的两个扣除cDNA文库 .从鼻咽癌相关的扣除cDNA文库中随机挑取 1 2 0 0个克隆 ,采用菌落PCR扩增其插入cDNA片段 ,自动点膜制备成cDNA微阵列膜 ,分别用鼻咽癌上皮细胞株HNE1、人胚鼻咽上皮mRNA经逆转录标记cDNA探针 ,分别与cDNA微阵列膜杂交 ,通过杂交信号的自动扫描分析 ,对杂交信号存在 5倍差异的克隆进行测序 ,获得了 1 0个鼻咽癌差异表达基因的cDNA片段 ,其中 3个为新基因序列 ,其GenBank登录号为 :AF5 1 0 1 88、AF5 1 0 1 89和AF5 1 0 1 90 ,7个代表已知基因序列 .采用RT PCR证实S1 0 0A8,CK1 9和RBP1基因在人胚鼻咽上皮中高表达而在鼻咽癌细胞株HNE1中低表达 .这些结果显示上述基因可能是鼻咽癌发生的重要因素     

Identification of symbiosis-regulated genes in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza by differential hybridization of arrayed cDNAs

Ectomycorrhiza development alters gene expression in the fungal and plant symbionts. The identification of a large number of genes expressed exclusively or predominantly in the symbiosis will contribute greatly to the understanding of the development of the ectomycorrhizal symbiosis. We have constructed a cDNA library of 4-day-old Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza and sequenced 850 cDNAs cloned randomly or obtained through suppression subtractive hybridization (SSH). Based on the absence of a database match, 43% of the ectomycorrhiza ESTs are coding for novel genes. At the developmental stage analysed (fungal sheath formation), the majority of the identified sequences represented 'housekeeping' proteins, i.e. proteins involved in gene/protein expression, cell-wall proteins, metabolic enzymes, and components of signalling systems. We screened arrayed cDNAs to identify symbiosis-regulated genes by using differential hybridization. Comparisons of signals from free-living partners and symbiotic tissues revealed significant differences in expression levels (differential expression ratio >2.5) for 17% of the genes analysed. No ectomycorrhiza-specific gene was detected. The results successfully demonstrate the use of the cDNA array and SSH systems as general approaches for dissecting symbiosis development, and provide the first global picture of the cellular functions operating in ectomycorrhiza.