A rapid screen for putative mislocalization mutations by using a GAL4-hybrid protein.

A GAL4 one-hybrid system was used to develop an indirect assay for intracellular localization. The Kar1 protein, a component of the yeast spindle pole body (SPB), was shown to be targeted to the SPB by electron microscopic analysis, or indirect immunofluorescence methods. To assay localization of the Kar1p by measuring the reporter gene expression on solid media, we constructed Kar1-Gal4 hybrid proteins with or without the SPB localization domain. The long fusion Kar1(299)-Gal4 with the localization domain led to non-growth on SC-His + AT media, and quite a low level of the lacZ reporter gene expression. The short fusion Kar1(107)-Gal4 without the localization domain caused a full activation of the reporter genes, HIS3 and lacZ, indicating that the protein remains in the nucleoplasm. These results suggest that the Kar1(299)-Gal4p fusion protein, localized to the SPB, can be clearly differentiated with the mislocalized protein by assaying the reporter gene expression. By utilizing the Kar1(299)-Gal4 construct, we isolated ten spontaneous mutations that were defective in the Kar1-Gal4p localization. Four of these mutations were in the same complementation group. We propose, therefore, that the Gal4-hybrid localization assay can be utilized in cases where the target organelle or the structure is too small for microscopic analysis, or in the initial screening for mutations defective in localization.     

The microcell-mediated transfer of human chromosome 8 restores the deficient N-acetylytransferase activity in skin fibroblasts of Mucopolysaccharidosis type IIIC patients

The candidate gene for Mucopolysaccharidosis (MPS) type IIIC has been localized to the pericentric region of the chromosome 8 by the linkage disequilibrium analysis. To validate the localization of the gene, we rescued the deficient acetyl-coenzyme A: alpha-glucosaminide-N-acetylytransferase activity in the cultured cells of MPS IIIC patients by functional complementation via microcell-mediated chromosome transfer. The introduction of the target human monochromosome completely restored the activity confirming functional localization of the candidate gene on human chromosome 8.     

人类生殖相关新基因的定位和组织表达

基因定位对研究基因之间以及基因与疾病之间的相互关系具有重要意义。应用辐射杂种细胞系技术（RH）对我们克隆的人类新基因HBRP（Human BSP-Related Protein）进行了染色体定位,结果将该基因定位于19q13.2～13.3,同时应用生物信息学方法在人类基因组重叠片段数据库进行该基因的定位,结果相吻合。研究证明,RH技术具有快速、精确、简便等优点,是基因定位研究中一强有力的技术。同时通过RT-PCR方法研究了HBRP基因在人体各组织中的表达分布,结果显示该基因在睾丸、肠、肾、肝、脾、胃、胰腺组织有较高的表达,而在检测的脑、肺、骨骼肌、心肌组织中表达较弱。 Abstract:Gene localization is significant in elucidating the interaction between genes,gene and diseases.Using radiation hybrid (RH) technique,we cloned and localized a novel gene,designated human BSP-related protein (HBRP) on 19q13.2～13.3,in line with its localization in data bank of overlapping fragment of human genome through bioinformatics method.It is suggested RH is rapid,precise,simple and powerful in gene localization.In addition,we detected the expression and distribution of HBRP in human tissues by RT-PCR.The results showed HBRP was highly expressed in intestine,kidney,liver,spleen,stomach and pancreas,whereas lowly in brain,lung,muscle and heart.     

基于遗传算法的基因芯片图像网格定位

对基因芯片荧光图像进行网格定位是进行芯片分析的前提与关键。利用为形模板匹配方法,基因芯片的自动网格定位问题转化为优化问题,从而用遗传算法求解。遗传算法是一种模拟自然界进化过程的启发式优化计算法,具高效及可收敛到全局最优的特点。实验证明该方法具有良好的准确性与可靠性。     

The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs

We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth.     

地高辛标记探针用于人白细胞基因定位

用地高辛标记探针在人染色体上进行了基因定位。使用了酶显色和荧光显色,两者得到了相同的定位结果,特异区阳性率分别为11.6％和19.8％’荧光显色特异性较高,说明基因定位效果受显示系统效率的影响,地高辛标记探针用于基因定位有比放射性探针、生物素探针更多的优越性。又讨论了几个影响效果的因素,提出以SDC代替甲酰胺洗涤;紫外照射于杂交前R显带方法能取得较好的基因定位效果。     

原核表达系统真核化中的核定位信号对乙肝病毒(HBV)基因免疫效果的影响

研究核定位信号对真核化的T7表达系统在真核细胞及基因免疫中表达HBsAg基因效率的影响。结果发现,在体外培养细胞中,无核定位信号的T7系统表达效率比有核定位信号组明显要高。基因免疫时,无核定位信号组诱发小鼠产生了针对HBsAg 的特异性免疫应答,而含核定位信号的T7表达系统则未能诱发小鼠发生明显的免疫应答。提示T7系统是一种胞浆表达系统,将T7RNA聚合酶引入核定位信号后该系统的表达效率降低,甚至不能诱发特异性免疫应答。     

Enhanced nuclear import and transfection efficiency of TAT peptide-based gene delivery systems modified by additional nuclear localization signals

Cellular uptake and nuclear localization are two major barriers in gene delivery. In order to evaluate whether additional nuclear localization signals (NLSs) can improve gene transfection efficiency, we introduced different kinds of NLSs to TAT-based gene delivery systems to form three kinds of complexes, including TAT-PV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1. The DNA binding ability of different vectors was evaluated by agarose gel electrophoresis. The in vitro transfections mediated by different complexes under different conditions were carried out. The cells treated by different complexes were observed by confocal microscopy. The MTT assay showed that all complexes did not exhibit apparent cytotoxicity in both HeLa and Cos7 cell lines even at high N/P ratios. The luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. Confocal study showed that, except TAT/DNA/PV, all other complexes exhibited enhanced nuclear accumulation and cellular uptake in both HeLa and Cos7 cell lines. These results indicated that the introduction of nuclear localization signals could enhance the transfection efficacy of TAT-based peptides, implying that the TAT peptide-based vectors demonstrated here have promising potential in gene delivery.     

Arginine-rich regions succeeding the nuclear localization region of the herpes simplex virus type 1 regulatory protein ICP27 are required for efficient nuclear localization and late gene expression.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein that localizes to the nuclei of infected cells. The strong nuclear localization signal (NLS) of ICP27 was identified recently and shown to reside in the amino-terminal portion of the polypeptide from residues 110 to 137 (W.E. Mears, V. Lam, and S.A. Rice, J. Virol. 69:935-947, 1995). There are also two arginine-rich regions directly succeeding the NLS. The first of these arginine-rich sequences (residues 141 to 151), together with the NLS, has been shown by Mears et al. to form the nucleolar localization signal. Arginine-rich motifs are common in domains involved in nuclear localization and RNA binding. To analyze the role of the arginine-rich regions in ICP27, we constructed stably transformed cell lines containing ICP27 mutants with deletions of all or parts of the NLS and arginine-rich regions. We also constructed mutants in which these regions were replaced with heterologous NLSs or RNA-binding domains. Characterization of these mutants indicated that the arginine-rich regions were required but not sufficient for wild-type localization of ICP27. More importantly, the NLS and arginine-rich regions were also essential to the function of ICP27. Mutants lacking these sequences were defective in late gene expression during infection even when ICP27 was properly localized to the nucleus by substitution of the NLS from simian virus 40 large T antigen. Further, the defect in late gene expression could not be overcome by replacement with the highly basic RNA-binding domain of human immunodeficiency virus type 1 Tat. The deficiency in late gene expression was independent of ICP27's role in stimulating viral DNA replication. In addition, localization of the HSV-1 proteins ICP4, ICP0, and ICP8 was unaffected by ICP27 mutants in this region. These results suggest that the arginine-rich regions are required for efficient nuclear localization and for the regulatory activity of ICP27 involved in viral late gene expression.     

人CDC73基因编码蛋白的理化性质及功能研究

采用生物信息学方法对人CDC73 (cell division cycle 73)基因编码蛋白的理化性质、亲疏水性、跨膜区域、信号肽区域、二级结构、三级结构、蛋白质之间的相互作用、亚细胞定位进行预测分析。使用多种分析软件对人CDC73基因编码蛋白进行预测分析。研究可知,人CDC73基因属于抑癌基因,该基因编码一个由531个氨基酸组成的肿瘤抑制因子Parafibromin,其等电点为9.63,半衰期为30 h且在哺乳动物中高度保守;二级结构预测发现13个α螺旋和10个β折叠片层,三级结构预测结果的可靠性达69.01%,亚细胞定位主要分布于细胞质及细胞核。由本研究可知,人CDC73基因编码蛋白是一个存在核定位序列的不稳定亲水蛋白,在细胞内广泛分布并参与多种生命活动过程,且能够抑制肿瘤的产生。对人CDC73基因编码蛋白结构和功能的预测分析,可为其进一步的研究提供一定的理论依据,也为相关疾病的诊治提供新的思路。     

Distal protein sequences can affect the function of a nuclear localization signal.

The major DNA-binding protein, or infected-cell protein 8 (ICP8), encoded by herpes simplex virus can localize to the cell nucleus independently of other viral proteins. To define the nuclear localization signals within ICP8, we performed several forms of mutagenesis on the cloned ICP8 gene. Deletion analysis of the ICP8 gene showed that several portions of ICP8 are involved in its nuclear localization. To determine whether these regions were independent localization signals, we introduced various portions of the ICP8 gene into a series of cassette plasmids which allowed expression of fusion proteins containing pyruvate kinase, normally a cytoplasmic protein, fused to various portions of ICP8. These results showed that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of targeting protein kinase into the nucleus. However, inclusion of certain additional regions of ICP8 into the fusion protein led to an inhibition of nuclear localization. Therefore, the carboxyl-terminal 28 residues of ICP8 can act independently as a nuclear localization signal, but certain conformational constraints or folding or assembly requirements in the remainder of the protein can affect the nuclear localization of the protein. Our results demonstrate that sequences distant from a nuclear localization signal can affect its ability to function. A set of fusion vectors has been isolated which should be of general use for making 5' or 3' fusions in any reading frame to rapidly map localization signals.     

Chromosomal localization of the mouse gene coding for the 68 kDa neurofilament subunit

The chromosomal localization of the mouse gene coding for the 68 kDa intermediate filament subunit of neurones (NF-L) was determined by in situ hybridization using specific 3H-labelled DNA probes. There is only one copy of the NF-L gene. The gene encoding NF-L is located on chromosome 14 region (D1-E1).     

Localization of hepatocyte growth factor (HGF) gene on human chromosome 7.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and a variety of epithelial cells in culture. The cDNAs for human and rat HGF have been cloned by different researchers, including ourselves; however, no information on the genomic structure and chromosome localization of the HGF gene is yet available. To investigate HGF's chromosomal localization, DNA from a battery of human-hamster somatic cell hybrids was digested with BglII and analyzed by Southern blot using a 2.3-kb human HGF cDNA as a hybridization probe. The gene encoding the human HGF was assigned to human chromosome 7. Restriction enzyme and Southern blot analyses using the HGF cDNA and HGF-specific oligonucleotides as probes suggest that the human HGF gene exists as a single-copy gene and is composed of several exons.     

Regional localization of the interferon-beta 2/B-cell stimulatory factor 2/hepatocyte stimulating factor gene to human chromosome 7p15-p21

The human interferon-beta 2 gene (IFNB2) is identical to the genes encoding the B-cell stimulatory factor (BSF-2), the hybridoma growth factor (HGF), and the hepatocyte stimulating factor (HSF). This protein mediates major alterations in the secretion of a wide spectrum of plasma proteins by the liver in response to tissue injury (the acute-phase response). We have used a cDNA probe specific to the human IFNB2 gene in DNA hybridization experiments and report the regional localization of this gene to human chromosome 7p15-p21. Southern blot analyses of DNA extracted from a panel of mouse X human somatic cell hybrids localized this gene to human chromosome 7p. In situ hybridization of the IFNB2 cDNA probe to prebanded human metaphase chromosome spreads allowed the further localization of this gene to 7p15-p21.     

Refined Localization of the α1-Subunit of the Skeletal Muscle L-Type Voltage-Dependent Calcium Channel (CACNL1A3) to Human Chromosome 1q32 by in Situ Hybridization

We isolated and partially sequenced a cosmid clone containing the human skeletal muscle L-type voltage-dependent calcium channel gene (CACNL1A3). The cosmid clone, which was also found to contain a novel dinucleotide repeat marker for the CACNL1A3 gene, was used for the chromosomal localization of CACNL1A3 by in situ hybridization. Our results refine the localization of CACNL1A3 on the long arm of human chromosome 1 to band q32.     

Factor XI gene (F11) is located on the distal end of the long arm of human chromosome 4

Chromosomal localization of the gene for human coagulation factor XI (F11) was determined by in situ hybridization using a genomic DNA probe which contained exons VIII, IX, and X of the gene. The results indicate that the gene is located at 4q35.     

酵母蛋白质相互作用与基因表达谱和亚细胞定位的相关性

研究酵母（yeast）蛋白质相互作用与基因表达谱和蛋白质亚细胞定位的关系.首先,构建了蛋白质相互作用正样本集、负样本集、随机组对负样本集和混合样本集.然后,对于4个数据集中的所有蛋白质对,通过比较它们的基于距离的基因共表达的分布以及它们中具有已知亚细胞定位的蛋白质对的共定位出现率,实现了这些高通量数据的交叉量化分析.结果揭示,与非相互作用蛋白质对相比,相互作用蛋白质对的基因表达谱具有较高的相似性;相互作用蛋白质对更倾向于具有相同的亚细胞定位.结果还揭示出这些蛋白质特征相关的总体趋势.     

Localization of the human erbB-2 gene on normal and rearranged chromosomes 17 to bands q12-21.32

Through the use of a cDNA probe, the human erbB-2 gene was localized by in situ hybridization of normal human chromosomes at 17q11-q21. In situ hybridization of chromosomes derived from fibroblasts carrying a constitutional 15;17t(q22.3;q11.21) translocation showed that the erbB-2 gene was relocated on the rearranged chromosome 15. These results as well as grain localization on prophase chromosomes locate the erbB-2 gene at 17q12-q21.32. This localization may facilitate the search for human malignancies with chromosome changes involving the erbB-2 gene.