A potential animal model for Lesch-Nyhan syndrome through introduction of HPRT mutations into mice

The human Lesch-Nyhan syndrome is a rare neurological and behavioural disorder, affecting only males, which is caused by an inherited deficiency in the level of activity of the purine salvage enzyme hypoxanthine-guanosine phosphoribosyl transferase (HPRT). How the resulting alterations in purine metabolism lead to the severe symptoms characteristic of Lesch-Nyhan patients is still not understood. No mutations at the Hprt locus leading to loss of activity have been described in laboratory animals. To derive an animal model for the Lesch-Nyhan syndrome, we have used cultured mouse embryonic stem cells, mutagenized by retroviral insertion and selected for loss of HPRT activity, to construct chimaeric mice. Two clonal lines carrying different mutant Hprt alleles have given rise to germ cells in chimaeras, allowing the derivation of strains of mutant mice having the same biochemical defect as Lesch-Nyhan patients. Male mice carrying the mutant alleles are viable and analysis of their cells shows a total lack of HPRT activity.     

HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells

Embryonal stem (ES) cell lines, established in culture from peri-implantation mouse blastocysts, can colonize both the somatic and germ-cell lineages of chimaeric mice following injection into host blastocysts. Recently, ES cells with multiple integrations of retroviral sequences have been used to introduce these sequences into the germ-line of chimaeric mice, demonstrating an alternative to the microinjection of fertilized eggs for the production of transgenic mice. However, the properties of ES cells raise a unique possibility: that of using the techniques of somatic cell genetics to select cells with genetic modifications such as recessive mutations, and of introducing these mutations into the mouse germ line. Here we report the realization of this possibility by the selection in vitro of variant ES cells deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT; EC 2.4.2.8), their use to produce germline chimaeras resulting in female offspring heterozygous for HPRT-deficiency, and the generation of HPRT-deficient preimplantation embryos from these females. In human males, HPRT deficiency causes Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation.     

猪色素上皮衍生因子的电子克隆及序列分析

用鼠的色素上皮衍生因子cDNA序列在猪的ESTs库进行BLASTn搜索,得到一系列不同大小的ESTs片段,经拼接得到完整cDNA序列.序列分析显示该cDNA长1 425 bp,有一个1 242 bp的开放阅读框,编码413个氨基酸,5’非编码区长53 bp,3’非编码区长130 bp,有一个加尾序列和多聚腺苷酸尾巴.其核苷酸序列与牛、人和鼠的同源性分别为89%、87%和82%,氨基酸的同源性分别为89%、87%和84%,且有保守的糖基化位点、半胱氨酸位点和serp in基序,说明所克隆的cDNA序列为猪的PEDF全长cDNA.     

Analysis of human T-lymphotrophic virus sequences in multiple sclerosis tissue

Several observations suggest that retroviral infection is involved in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). First, lymphadenopathy-associated virus/human T-lymphotropic virus type III (LAV/HTLV-III), the agent of acquired immune deficiency syndrome (AIDS), has been shown to be neurotropic in man. Second, the genetic organization of the lentivirus visna, which causes a chronic demyelinating disease of sheep, closely resembles that of LAV/HTLV-III. Recently, Koprowski and colleagues reported that MS is associated both with raised levels of circulating antibodies to HTLV-I and with the presence of HTLV-I-specific RNA within cell lines derived from the cerebrospinal fluid (CSF). Here we report that no HTLV-I-like or LAV/HTLV-III-like sequences can be detected, by in situ hybridization, in central nervous system (CNS) tissues from MS patients, and that nonspecific HTLV-I-like signal in peripheral blood mononuclear cells or in CSF cell lines is characteristic of MS. Furthermore, enzyme-linked immunosorbent assay (ELISA) analysis of circulating and CSF antibodies for HTLV-I reactivity fails to distinguish between MS and control groups.     

Cloning of human cytokine signal transduction inhibitor genehumSOCS-2

An EST (gb/AA115239) with high identity to the mouse cytokine signal transduction inhibitor genemmSOCS-2 was selected in GenBank EST database by the homologous screening method. The cDNA with the same sequence of the EST was got in human placenta cDNA library by PCR and a 1011 bp cDNA fragment was selected using above cDNA as probes to perform walking hybridization in placenta cDNA library. The cDNA fragment contains one 594 bp open reading frame (ORF) which encodes 198 amino acid residues. It was proved to be novel after NCBl database screening. Homology comparison showed that this gene has 93% identity tommSOCS-2 at the amino acid level and it has high identities to other related genes in SH₂ domain and SOCS box, so it was namedhumSOCS-2 and the accession number in GenBank is gb/AF020590. The expression analysis showed that the gene is expressed obviously higher in prostate than in other 15 human tissues.     

A gene expression atlas of the central nervous system based on bacterial artificial chromosomes

The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.     

BMP－3及BMP－5在不同组织和细胞中的表达

报道ＢＭＰ－３及ＢＭＰ－５在不同组织细胞中的表达．将人的神经母细胞瘤ＳＫ细胞的总ＲＮＡ及人的脑、肝、胸腺、脾、胎盘及睾丸的总ＲＮＡ反转录成ｃＤＮＡ作为模板，利用设计的编码ＢＭＰ－３和ＢＭＰ－５成熟蛋白的专一性引物分别扩增出相应的片段．ＰＣＲ产物的琼脂糖凝胶电泳结果表明，ＢＭＰ－３及ＢＭＰ－５在不同组织细胞中表达类型不同．     

Expression of human adenosine deaminase in murine haematopoietic progenitor cells following retroviral transfer

Adenosine deaminase (ADA) deficiency, an autosomal recessive inborn error of metabolism, leads to severe combined immune deficiency in man. This enzyme, although constitutively expressed in most tissues, is expressed at high level in immature T cells, and study of the pathophysiology of the disorder indicates that increased deoxyadenosine or altered methylation capacity have toxic effects on T-cell maturation. Although bone marrow transplantation can correct the immune deficiency, this therapy is associated with graft-versus-host disease and incomplete immune restoration, and so our laboratory and others have sought to develop a method of gene replacement as a possible treatment for the disease. Moreover, characterization of the complementary DNA of the human ADA gene and some of its mutants makes it possible to design gene transfer strategies. We have now subcloned a human adenosine deaminase cDNA into the retrovirus shuttle vector pZIP-SV(B), and in this way have isolated a cell line, 4.2T, which produces high titres of replication-defective retrovirus which have been used to transfer the gene for human ADA to mouse bone marrow cells. Transfer and expression of the neomycin-resistance gene (neo) and the ADA gene in murine bone marrow colony-forming units (CFU) was demonstrated by in vitro colony formation in the presence of the antibiotic G418 or 9-xylofuranosyladenine plus deoxycoformycin, respectively. Isoenzyme analysis also showed human ADA expression in the cultured mouse bone marrow.     

Cloning and expression analysis of MBLL cDNA

The mbl (muscleblind) gene of Drosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila's photoreceptors. Primers have been designed according to human EST086139, which is highly homologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLL is a widely expressed gene, but the expression amounts differ in these tissues.     

生物信息学技术克隆并分析新基因STRF7

为进一步研究信号转导相关的新基因片段BE644250,采用生物信息学方法克隆基全长cDNA,并分析了其ORF,电子表达谱,染色体定位等,之后对全长序列进行了实验验证。电子延伸（contig)获得了729bp的延伸产物,含一个典型的74aa的ORF,命名为STRF7。与已知蛋白无明显同源性,部分地相似于人的源框蛋白CDX－4和酵母的转录调节子ADR6,属一新发现的基因;RT－PCR从IL－6刺激后的U937中克隆了STRF7基因,基序列与电子延伸结果安全一致,进一步的分析显示STRF7在多种组织中表达并定位于第6号染色体上,上述结果显示,STRF7是一个新基因,编码含74aa的蛋白,并且是一个潜在的转录因子。     

Comparison of transformation efficiency of human active and inactive X-chromosomal DNA

The mechanism of X-chromosome inactivation has been investigated recently using DNA-mediated transformation of the X-linked hypoxanthine phosphoribosyl transferase (hprt) locus. Several experiments indicate that inactive X-chromosomal DNA does not function in HPRT transformation. Liskay and Evans used DNA from hamster or mouse cells which had an hprt- allele on the active X chromosome and an hprt+ allele on the inactive X chromosome. We and others used rodent-human hybrid cell lines which had an hprt+ allele on the inactive human X chromosome alone. DNA from all of these cells failed to transform HPRT- recipients. Recently, Chapman et al. have shown that inactive X-chromosome DNA from several tissues of adult female mice is strikingly inefficient in genetic transformation for the hprt gene. On the other hand, de Jonge et al., using simian virus 40 (SV40)-transformed fibroblasts from a human heterozygous for an HPRT deficiency, observed HPRT transformation regardless of whether the hprt+ allele was on the active or the inactive X chromosome of the donor cells. We have done an experiment similar to that of deJonge et al., and report here results which clearly indicate that DNA from the inactive X chromosome functions very poorly in HPRT transformation, thus supporting the original interpretation of Liskay and Evans that inactive X-chromosomal DNA is structurally modified.     

人类磷酸泛酰巯基乙胺结合蛋白新基因的分离和鉴定

在对AD293和HEK293进行差减杂交以探索两者在吸附和凋亡特性上的差异时,从AD293的高表达文库中分离得到一段新的cDNA片段.从人类胎脑文库克隆得到该基因,全长2 745 bp,编码的蛋白含518个氨基酸,被预测为磷酸泛酰巯基乙胺结合蛋白.该基因在染色体上定位于2p22.3,包含8个外显子.该cDNA编码的蛋白序列含有一个凋亡抑制蛋白5结构域,外皮蛋白重复片段和铜结合辛肽重复片段.RT-PCR分析显示该基因在人类正常组织和癌组织中广泛表达,但在癌组织中表达量相对较低,提示其可能对细胞凋亡有抑制作用.该基因在进化过程中高度保守.     

Pericytes regulate the blood-brain barrier

The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB.     

从EST出发克隆人基因组Xq13区段一个候选基因的初步研究

人类基因组表达序列筛选是寻找候选基因的重要路线之一，外显子陷阱法，ｃＤＮＡ直接筛筛选法，它们可分别根据表达序列的结构及表达特点进行筛选，ＥＳＴ是表达图的位标，它们是一些位点专一的表达序列位标，根据ＥＳＴ的特征，在国内首次建立了一种从ＥＳＴ出发的筛选候选基因的新方法，用睦方法已在人Ｘ染色体Ｘｑ１３区段筛选得到了一个新的ｃＤＮＡ，总测序徇的１３９８ｂｐ包含了完整的３末端。     

猪Akt1、Akt2基因克隆与组织表达图谱分析

为了得到长白猪蛋白激酶Akt1和Akt2基因序列并分析其表达模式,本研究使用RT-PCR方法,首先克隆了蛋白激酶Akt1和Akt2的cDNA.序列分析显示:长白猪Akt1基因的cDNA全长1461bp,编码具480个氨基酸残基的前体蛋白,其氨基酸序列与人,牛,大鼠,小鼠同源性达到97%以上.长白猪Akt2基因cDNA全长为1505bp,编码具481个氨基酸残基的前体蛋白,其氨基酸序列与人,牛,大鼠,小鼠的同源性高达97%以上.SMART分析表明,猪Akt1和Akt2蛋白均包含了与PI-3K结合的PH结构域及2个具有丝氨酸/苏氨酸激酶催化活性的S_TKc结构域.RT-PCR检测结果显示:Akt1mRNA在垂体、心脏、肝脏、脾脏、肌肉组织中高表达,在大脑、小脑、肾脏表达丰度较低.Akt2则在小脑、垂体、心脏、肝脏、脾脏和肌肉组织中高表达,而在大脑和肾脏中表达丰度较低.     

胚胎性横纹肌肉瘤中下调表达基因HumCyr61的分？…

分离和克隆胚胎性横纹肌肉瘤中下调表达基因ＨｕｍＣｙｒ６１。方法：利用计算机辅助的同源扩增策略，成功克隆一个长１８８７ｂｐ ｃＤＮＡ，并与ＧｅｎＢａｎｋ核酸数据库比较。结果：该基因与鼠生长因子诱导早期表达的Ｃｙｒ６１基因同源度为８２％，在蛋白质水平上的同源度高达９２％，其中对高级结构有重大贡献的半胱氨酸和脯氨酸等氨基酸残基高度保守。查新结果还显示，它包括在胚胎性横纹肌肉瘤细胞系ＲＤ中表达呈下调表达的