Nesprins: Tissue-Specific Expression of Epsilon and Other Short Isoforms

Nesprin-1-giant and nesprin-2-giant regulate nuclear positioning by the interaction of their C-terminal KASH domains with nuclear membrane SUN proteins and their N-terminal calponin-homology domains with cytoskeletal actin. A number of short isoforms lacking the actin-binding domains are produced by internal promotion. We have evaluated the significance of these shorter isoforms using quantitative RT-PCR and western blotting with site-specific monoclonal antibodies. Within a complete map of nesprin isoforms, we describe two novel nesprin-2 epsilon isoforms for the first time. Epsilon isoforms are similar in size and structure to nesprin-1-alpha. Expression of nesprin isoforms was highly tissue-dependent. Nesprin-2-epsilon-1 was found in early embryonic cells, while nesprin-2-epsilon-2 was present in heart and other adult tissues, but not skeletal muscle. Some cell lines lack shorter isoforms and express only one of the two nesprin genes, suggesting that either of the giant nesprins is sufficient for basic cell functions. For the first time, localisation of endogenous nesprin away from the nuclear membrane was shown in cells where removal of the KASH domain by alternative splicing occurs. By distinguishing between degradation products and true isoforms on western blots, it was found that previously-described beta and gamma isoforms are expressed either at only low levels or with a limited tissue distribution. Two of the shortest alpha isoforms, nesprin-1-alpha-2 and nesprin-2-alpha-1, were found almost exclusively in cardiac and skeletal muscle and a highly conserved and alternatively-spliced exon, available in both nesprin genes, was always included in these tissues. These “muscle-specific” isoforms are thought to form a complex with emerin and lamin A/C at the inner nuclear membrane and mutations in all three proteins cause Emery-Dreifuss muscular dystrophy and/or inherited dilated cardiomyopathy, disorders in which only skeletal muscle and/or heart are affected.     

Identification and Functional Analysis of Three Isoforms of Bovine BST-2

Human BST-2 (hBST-2) has been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. Orthologues of BST-2 have been identified in several species, including human, monkeys, pig, mouse, cat and sheep. All have been reported to possess antiviral activity. Duplication of the BST-2 gene has been observed in sheep and the paralogues are referred to as ovine BST-2A and BST2-B, although only a single gene corresponding to BST-2 has been identified in most species. In this study, we identified three isoforms of bovine BST-2, named bBST-2A1, bBST-2A2 and bBST-2B, in bovine cells treated with type I interferon, but not in untreated cells. Both bBST-2A1 and bBST-2A2 are posttranslationally modified by N-linked glycosylation and a GPI-anchor as well as hBST-2, while bBST-2B has neither of these modifications. Exogenous expression of bBST-2A1 or bBST-2A2 markedly reduced the production of bovine leukemia virus and vesicular stomatitis virus from cells, while the antiviral activity of bBST-2B was much weaker than those of bBST-2A1 and bBST-2A2. Our data suggest that bBST-2A1 and bBST-2A2 function as part of IFN-induced innate immunity against virus infection. On the other hand, bBST-2B may have a different physiological function from bBST-2A1 and bBST-2A2.     

Tissue-Specific Isoforms of Catalase Subunits in Castor Bean Seedlings

Catalases purified from endosperm glyoxysomes and non-specializedmicrobodies from hypocotyls of castor bean seedlings differedin their specific activity [90–164 and 0.89–4.9kunits (mg protein)^–1, respectively] and in their constituentsubunits [two subunits of 54 and 56 kDa for the endosperm enzymeand only one of 56 kDa for the hypocotyl enzyme]. Immunoblotanalysis also showed that particulate fractions from the endospermsand from etiolated and green cotyledons contained two catalasesubunits of 54 and 56 kDa, whereas such fractions from the hypocotylsand roots contained only the 56-kDa subunit. Leaf peroxisomesfrom green leaves had two catalase subunits of around 55 kDaeach. Results of translation in vitro indicated that the 54-and 56-kDa subunits were translated from distinct mRNAs andlevels of both mRNAs increased in the endosperms during germination,prior to increases in levels of catalase proteins. In the hypocotyls,the 56-kDa subunit seemed to be synthesized constitutively. ¹Present addresses: YO, Toyota Central Institute, 31-9 Musashizuka,Nagabuchi, Nagakute, Aichi 480-11, Japan     

The Tissue-Specific Gene Expression during Progression of Mouse Hepatocellular Carcinoma

Expression of hepato-specific genes in slow- and fast-growing hepatocellular murine carcinomas was studied. A fast-growing dedifferentiated transplantable hepatocarcinoma variant (fgHCC) arose from the highly differentiated slow-growing hepatocarcinoma (sgHCC). In contrast to the parental hepatocarcinoma, expression of the hepatocyte nuclear factor 4 (HNF4), one of the key regulators of hepatocyte differentiation, and several HNF-4-responsive genes, transferrin, transthyretin, hepatocyte nuclear factor 1 (HNF1), and serum albumin, was downregulated in fgHCC. The expression of exogenous HNF4 in the fgHCC cell culture partially restored the expression of hepato-specific genes and led to the formation of epithelial islets in the culture. The described system may serve as an appropriate model for further analysis of mechanisms underlying hepatocarcinogenesis and liver tumor progression.     

Bayesian Joint Analysis of Gene Expression Data and Gene Functional Annotations

Identifying which genes and which gene sets are differentially expressed (DE) under two experimental conditions are both key questions in microarray analysis. Although closely related and seemingly similar, they cannot replace each other, due to their own importance and merits in scientific discoveries. Existing approaches have been developed to address only one of the two questions. Further, most of the methods for detecting DE genes purely rely on gene expression analysis, without using the information about gene functional grouping. Methods for detecting altered gene sets often use a two-step procedure, of which the first step conducts differential expression analysis using expression data only, and the second step takes results from the first step and tries to examine whether each predefined gene set is overrepresented by DE genes through some testing procedure. Such a sequential manner in analysis might cause information loss by just focusing on summary results without using the entire expression data in the second step. Here, we propose a Bayesian joint modeling approach to address the two key questions in parallel, which incorporates the information of functional annotations into expression data analysis and meanwhile infer the enrichment of functional groups. Simulation results and analysis of experimental data obtained for E.?coli show improved statistical power of our integrated approach in both identifying DE genes and altered gene sets, when compared to conventional methods.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

口蹄疫病毒3D基因的克隆表达及功能分析

利用RT-PCR技术扩增了口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因,并将其克隆到原核表达质粒载体pET-28a(+)中.3D基因经测序确认后在大肠杆菌BL-21中表达,表达产物纯化的目的蛋白进行Western-blotting检测,获得分子量约55KDa的单一3D基因表达产物.利用RNA体外复制体系和荧光定量PCR技术,证明纯化的3D基因表达产物RNA依赖的RNA聚合酶具有较高的酶活性,可以在体外从头合成FMDV RNA,且主要以引物依赖的方式合成病毒基因组.     

人锰超氧化物歧化酶基因剪接异构体的表达分析

对人锰超氧化物歧化酶(human manganese superoxide dismutase,hMn-SOD)基因剪接异构体进行分析,并检测异构体的表达情况。在GenBank库中检索人锰超氧化物歧化酶基因异构体及编码基因组序列,利用Vector NTI9生物软件进行核酸及蛋白序列比对;利用RT-PCR方法分析锰超氧化物歧化酶基因异构体的表达。结果显示,在GenBank库检索发现有3种人锰超氧化物歧化酶基因异构体,剪接异构的类型为可变的5′剪接位点和外显子盒,各异构体基因内含子均符合"GT-AG"规则。3种基因异构体编码两种异构体蛋白,即222个氨基酸的人锰超氧化物歧化酶蛋白以及中部缺少39个氨基酸的截短型异构蛋白。RT-PCR检测结果表明,剪接异构体hMn-SODb在HEK293T和HSC细胞中的表达比在HepG2细胞中高,未见异构体hMn-SODc的表达。     

豌豆肌动蛋白异型体的表达与系统发生分析

豌豆 (PisumsativumLinn .)肌动蛋白基因至少可以分为 3类异型体 :PEAcⅠ、PEAcⅡ和PEAcⅢ ,它们的编码区序列相似性很高 ,而非编码区序列存在显著的差异。RT_PCR和以 3′端非翻译区作探针的Southern杂交结果显示豌豆肌动蛋白异型体基因在根、茎、叶、卷须、花粉和幼嫩果实中均表达 ,但表达强度存在明显差异。利用连续稀释电泳对PEAcⅠ的转录本水平进行分析后发现它倾向于在幼嫩的器官中表达 ,且在 7d的茎中表达最强 ;在一个月内的叶片中表达也较强 ,此后明显下降 ;卷须中表达变化不显著 ,在花粉中表达很弱 ,在幼嫩荚果中的表达亦较多。PEAcⅡ表达特点与PEAcⅠ具有某些相似性。根据肌动蛋白序列重建的系统发生树表明豌豆肌动蛋白 3类异型体间的分歧明显 ,但起源于单、双子叶植物分化前的共同基因祖先。推测豌豆肌动蛋白异型体基因在转录调控和细胞功能上存在差异。     

豌豆肌动蛋白异型体的表达与系统发生分析

豌豆(Pisum sativum Linn.)肌动蛋白基因至少可以分为3类异型体: PEAcⅠ、PEAcⅡ和PEAcⅢ,它们的编码区序列相似性很高,而非编码区序列存在显著的差异.RT-PCR和以3′端非翻译区作探针的Southern杂交结果显示豌豆肌动蛋白异型体基因在根、茎、叶、卷须、花粉和幼嫩果实中均表达,但表达强度存在明显差异.利用连续稀释电泳对PEAcⅠ的转录本水平进行分析后发现它倾向于在幼嫩的器官中表达,且在7 d的茎中表达最强;在一个月内的叶片中表达也较强,此后明显下降;卷须中表达变化不显著,在花粉中表达很弱,在幼嫩荚果中的表达亦较多.PEAcⅡ表达特点与PEAcⅠ具有某些相似性.根据肌动蛋白序列重建的系统发生树表明豌豆肌动蛋白3类异型体间的分歧明显,但起源于单、双子叶植物分化前的共同基因祖先.推测豌豆肌动蛋白异型体基因在转录调控和细胞功能上存在差异.