Rb基因在小鼠胚胎发育中期肾组织的表达

用原位杂交法检测小鼠肾组织中细胞周期调控基因Rb的表达;用免疫组织化学及缺口末端标记法观察小鼠肾组织发育过程中的细胞增殖与凋亡：结果表明,Rb基因主要在肾上皮细胞表达,随胎龄增长而表达颜色加深。肾组织在第13天有一个较大的增殖增长,到第14天生长趋于稳定。以上结果说明Rb基因在小鼠胚胎肾发育中期有表达,该基因在胚胎肾发育中期起一定的作用;同时胚胎发育中期肾细胞增殖与凋亡相伴存在。     

Target Repression Induced by Endogenous microRNAs: Large Differences,Small Effects

MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity – that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.     

小鼠生精相关基因SRG4的cDNA克隆及在小鼠各发育阶段的表达

从大鼠精子尾部基因Spag4出发,在dbEST数据库中同源搜寻与大鼠Spag4基因编码氨基酸同源性较高的表达序列标签(Expressed Sequence Tag,EST),找到两个在小鼠精母细胞中表达的ESTs,BG101130和BG100990。通过电子杂交得到1155bp的片段,包含了小鼠假想基因AK006225的全部序列,定位于2H1-H2,其开放阅读框为87～1133bp,并被RT-PCR所证实,将该基因命名为小鼠生精相关基因4(Sperrnatogenesis Related Gene4,SRG4,GenBank登录号为AY307077)。SRG4基因推定编码348个氨基酸,有一个coiled-coil区,可能是一个跨膜蛋白。该基因与人类同源基因TSARG4同源性为74％(277／374),与大鼠Spag4同源性为45％(103／224)。多组织RT-PCR和Northern blot结果显示,SRG4在睾丸中特异性表达。RT-PCR结果发现,小鼠出生后2周内,SRG4表达量极低;3周开始时SRG4大量表达,到4∽5周时表达量最高。该结果提示。SRG4基因可能在小鼠精子形成过程中发挥着重要的作用。     

多个顺式作用元件调节血管紧张素原基因表达

血管紧张素原是最强的血管活性物质——血管紧张素Ⅱ的唯一前体,在不同的生理和病理条件下,其水平各异．为了研究血管紧张素原基因表达的调控,将人血管紧张素原基因５′端侧翼序列１．２ｋｂ同氯霉素乙酰转移酶（ＣＡＴ）基因编码序列连接,构成表达载体,并且在此基础上构建５′端系列缺失的突变表达载体,用这些表达载体转染ＨｅｐＧ２和ＣＯＳ－７,确定了正负调控元件;同时应用ＤＮＡ－蛋白质凝胶泳动检测技术,发现核蛋白质与该顺式元件的结合,从而证明多个顺式作用元件调节血管紧张素原基因的表达．     

Multiple Mechanisms Regulate Imprinting of the Mouse Distal Chromosome 7 Gene Cluster

Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57^Kip2, as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57^Kip2 during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57^Kip2, and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57^Kip2 have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57^Kip2 is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).     

Mastoparan-Induced Intracellular Ca2+ Fluxes May Regulate Cell-to-Cell Communication in Plants

The relationship of Ca2+ and plasmodesmatal closure was examined in staminal hairs of Setcreasea purpurea by microinjecting cells with active mastoparan (Mas-7), inactive mastoparan (Mas-17), active inositol-1,4,5-trisphosphate (IP3), or inactive IP3. Calcium green dextran 10,000 was used to study cellular free Ca2+, and carboxyfluorescein was used to monitor plasmodesmatal closure. When Mas-7 was microinjected into the cytoplasm of cell 1 (the tip cell of a chain of cells), a rapid increase in calcium green dextran-10,000 fluorescence was observed in the cytoplasmic areas on both sides of the plasmodesmata connecting cells 1 and 2 during the same time that the diffusion of carboxyfluorescein through them was blocked. The inhibition of cell-to-cell diffusion was transient, and the closed plasmodesmata reopened within 30 s. The elevated Ca2+ level near plasmodesmata was also transient and returned to base level in about 1.5 min. The transient increase in Ca2+, once initiated in cell 1, repeated with an oscillatory period of 3 min. Elevated Ca2+ and oscillations of Ca2+ were also observed near interconnecting cell walls throughout the chain of cells, indicating that the signal had been transmitted. Previously, we reported that IP3 closed plasmodesmata; now we report that it stimulated Ca2+ and oscillations similar to Mas-7. The effect was specific for similar concentrations of Mas-7 over Mas-17 and active IP3 over inactive IP3. It is important that the Ca2+ channel blocker La3+ eliminated the responses from Mas-7 and IP3, indicating that an influx of Ca2+ was required. These results support the contention that plasmodesmata functioning is regulated via Ca2+ and that IP3 may be an intermediary between the stimulus and Ca2+ elevations.     

Dicistronic Gene Expression in Developing Zebrafish

Internal ribosome entry sites (IRESs) allow ribosomal access to messenger RNA without a requirement for cap recognition and subsequent scanning to an initiator AUG. Hence, IRESs have been adapted into dicistronic vectors for the expression of more than one gene from a single mRNA. Dicistronic vectors have been used for many applications in mammalian tissue culture and transgenesis. However, whether the IRESs from mammalian viruses function without temporal or spatial restrictions in nonmammalian organisms like zebra fish (Danio rerio) is unknown. Therefore, we have examined the expression capabilities of the encephalomyocarditis virus (EMCV) IRES during zebrafish embryogenesis. We determined that the EMCV IRES was sufficient to permit detectable expression of several second cistron reporters during zebrafish embryogenesis, including luciferase and green fluorescent protein. This suggests that our dicistronic vectors are suitable for general use in any vertebrate system, from fish to humans. Received March 26, 1999; accepted June 14, 1999.     

Dynamics of ATP-induced Calcium Signaling in Single Mouse Thymocytes

Extracellular ATP (ATP_o) elicits a robust change in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in fura-2–loaded mouse thymocytes. Most thymocytes (60%) exposed to ATP_o exhibited a biphasic rise in [Ca²⁺]_i; [Ca²⁺]_i rose slowly at first to a mean value of 260 nM after 163 s and then increased rapidly to a peak level of 735 nM. In many cells, a declining plateau, which lasted for more than 10 min, followed the crest in [Ca²⁺]_i. Experiments performed in the absence of extracellular [Ca²⁺]_o abolished the rise in thymocyte [Ca²⁺]_i, indicating that Ca²⁺ influx, rather than the release of stored Ca²⁺, is stimulated by ATP_o. ATP_o- mediated Ca²⁺ influx was potentiated as the [Mg²⁺]_o was reduced, confirming that ATP⁴⁻ is the active agonist form. In the absence of Mg²⁺ _o, 3′-O-(4-benzoyl)benzoyl-ATP (BzATP) proved to be the most effective agonist of those tested. The rank order of potency for adenine nucleotides was BzATP⁴⁻>ATP⁴⁻>MgATP²⁻>ADP³⁻, suggesting purinoreceptors of the P2X₇/P2Z class mediate the ATP_o response. Phenotyping experiments illustrate that both immature (CD4⁻CD8⁻, CD4⁺CD8⁺) and mature (CD4⁺CD8⁻, CD4⁻CD8⁺) thymocyte populations respond to ATP. Further separation of the double-positive population by size revealed that the ATP_o-mediated [Ca²⁺]_i response was much more pronounced in large (actively dividing) than in small (terminally differentiated) CD4⁺CD8⁺ thymocytes. We conclude that thymocytes vary in sensitivity to ATP_o depending upon the degree of maturation and suggest that ATP_o may be involved in processes that control cellular differentiation within the thymus.Extracellular ATP (ATP_o)¹ and its metabolic products evoke physiological responses in virtually all tissues and cell types from central nervous to peripheral organ systems (for review see Dubyak and El-Moatassim, 1993; Harden et al., 1995). Tissues and isolated cells vary in sensitivity to purine agonists. Nucleotides (ATP, ADP, and AMP) and adenosine, the nucleoside product of ATP catabolism, elicit distinct responses in target cells by triggering P2 and P1 purinergic receptors, respectively (Burnstock, 1978). P2 purinoceptors can be further separated into two broad categories. The first group, divided into P2Y and P2U subtypes, couples nucleotide binding to effector molecules via G proteins. The second P2 category is comprised of nucleotide-sensitive ion channels and pores. ATP-gated P2 purinoceptors, designated P2X₁ through P2X₆ (cation channels) and P2X₇ (a dual function cation channel/pore), display extensive sequence identity (North, 1996) but disparate tissue distribution, biophysical properties, agonist profiles, and pharmacology (P2X₁, Valera et al., 1994; P2X₂-P2X₆, Collo et al., 1996; P2X₇, Surprenant et al., 1996). Moreover, P2X receptors functionally resemble acetylcholine- and serotonin-gated channels with respect to gating and ionic permeability but are structurally unique. Thus, nucleotides, together with acetylcholine, glutamate, GABA, glycine, and serotonin, are included in a small group of compounds that function as agonists for a structurally diverse set of ligand-gated ion channels and pores, as well as G protein-coupled receptors.ATP_o elicits a broad spectrum of physiological changes in cells of the immune system. In mast cells, ATP release has been shown to mediate cell-to-cell signaling (Osipchuk and Cahalan, 1992). In lymphocytes, ATP_o triggers cellular depolarization, greater permeability to small organic molecules (<400 D; Wiley et al., 1993; Chused et al., 1996), and a rise in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i; El-Moatassim et al., 1987; Wiley and Dubyak, 1989). The ATP_o-mediated rise in [Ca²⁺]_i modifies the functional properties of thymocytes via DNA synthesis (Gregory and Kern, 1978, 1981; Ikehara et al., 1981) and blastogenesis (El-Moatassim et al., 1987). Moreover, an increase in [Ca²⁺]_i has been linked to programmed cell death in thymocyte populations; Ca²⁺ release from intracellular stores evoked by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor, triggers the DNA fragmentation correlated with thymocyte apoptosis (Jiang et al., 1994; Zhivotovsky et al., 1994).Based upon a sensitivity profile for purine agonists and pharmacological agents, lymphocytes are not believed to possess G protein-linked purinoceptors (El-Moatassim et al., 1989b ). Rather, lymphocytes and related cell lines express purinoceptors of the ion channel/pore subtype (P2X₇). This ATP-gated pathway, originally termed P2Z (Gordon, 1986), has been characterized in mast cells (Cockcroft and Gomperts, 1979a ; Tatham and Lindau, 1990), transformed 3T3 fibroblasts (Heppel et al., 1985), macrophages (Buisman et al., 1988), parotid acinar cells (Soltoff et al., 1992), and phagocytic cells of the thymic reticulum (Coutinho-Silva et al., 1996). During whole cell patch–clamp experiments, putative P2Z channels in human B lymphocytes (Bretschneider et al., 1995) and rat peritoneal macrophages (Naumov et al., 1995) exhibit rapid activation kinetics when exposed to ATP_o. The ATP_o response depends critically upon extracellular divalent cations (Mg²⁺ and Ca²⁺), such that cellular depolarization and membrane permeability are greatest in divalent-free media. The ability of Mg²⁺- and Ca²⁺–ATP complexes to reduce receptor occupancy by lowering the concentration of ATP⁴⁻, the effective form of the nucleotide agonist, is a hallmark of P2X₇/P2Z purinoceptor physiology (Cockcroft and Gomperts, 1979b ).In this study, we examined the dynamics of [Ca²⁺]_i changes elicited by ATP_o at the single-cell level in fura-2– loaded thymocytes. To our surprise, we found that the ATP_o-mediated [Ca²⁺]_i increase varies significantly between individual cells. Moreover, the kinetics of the rise in [Ca²⁺]_i at the single-cell level is characterized by a biphasic time course that is not detectable in average profiles. To correlate stages of thymocyte development with the degree of sensitivity to ATP_o, we measured the surface expression of specific T-lymphocyte markers, CD4 and CD8, before performing Ca²⁺-imaging experiments. Our data illustrate that thymocytes vary in sensitivity to ATP_o depending upon level of maturation and degree of blastogenesis. Small, terminally differentiated, CD4⁺CD8⁺ thymocytes were least sensitive to ATP_o, while 90% of the single-positive (CD4⁺CD8⁻ or CD4⁻CD8⁺) cells, believed to be the immediate precursors of mature peripheral T-lymphocytes, exhibited a robust, ATP_o-dependent rise in [Ca²⁺]_i. The in vitro data we have gathered suggest that ATP_o may drive thymocyte differentiation in the intact thymus.     

Prediction of Associations between microRNAs and Gene Expression in Glioma Biology

Despite progress in the determination of miR interactions, their regulatory role in cancer is only beginning to be unraveled. Utilizing gene expression data from 27 glioblastoma samples we found that the mere knowledge of physical interactions between specific mRNAs and miRs can be used to determine associated regulatory interactions, allowing us to identify 626 associated interactions, involving 128 miRs that putatively modulate the expression of 246 mRNAs. Experimentally determining the expression of miRs, we found an over-representation of over(under)-expressed miRs with various predicted mRNA target sequences. Such significantly associated miRs that putatively bind over-expressed genes strongly tend to have binding sites nearby the 3'UTR of the corresponding mRNAs, suggesting that the presence of the miRs near the translation stop site may be a factor in their regulatory ability. Our analysis predicted a significant association between miR-128 and the protein kinase WEE1, which we subsequently validated experimentally by showing that the over-expression of the naturally under-expressed miR-128 in glioma cells resulted in the inhibition of WEE1 in glioblastoma cells.     

小鼠cAMP应答元件结合蛋白基因在大肠杆菌中的表达

采用PCR技术,删除了小鼠CREBcDNA5′端和3′端非编码序列,并引入便于基因操作的酶切位点。经30次循环的扩增,得到改造后的CREBcDNA,全长1071bp。亚克隆后,对此扩增片段进行了限制性内切酶物理图谱分析,测定了DNA序列,并以其为插入物,构建了pBV220-PCR-CREB重组表达载体。经Western印迹法分析证明,在大肠杆菌中的表达获得成功 。     

Gm2052基因在小鼠胚胎发育中表达的初步研究

目的:研究预测的编码蛋白基因Gm2052在小鼠胚胎发育阶段的表达模式,为进一步了解该基因的功能奠定基础。方法:通过全胚胎原位杂交技术、组织切片原位杂交技术及半定量RT-PCR方法,对预测的Gm2052基因在小鼠胚胎发育中后期及在新生小鼠中的表达情况进行初步分析。结果:全胚胎原位杂交显示,在E10.5小鼠胚胎中,Gm2052仅在脑中表达;当小鼠胚胎发育至E13.5时,Gm2052在脑、舌、肺、肝脏、胰腺等组织中均有表达。半定量RT-PCR结果显示,在小鼠胚胎中后期(E15.5和E18.5)及新生小鼠(出生后第9 d)中,Gm2052呈动态表达模式。结论:预测基因Gm2052与小鼠脑的发育密切相关,并可能参与小鼠肺、肝脏及胰腺等主要脏器胚期的发育。