Synthetic peptides as nuclear localization signals

The nuclear envelope defines a compartment boundary which is penetrated by pores that mediate a remarkable transport process. Precursor RNAs are retained in the nucleus, while processed messenger RNA, transfer RNA and ribosomal subunits are transported to the cytoplasm. Proteins destined for the nucleus become localized soon after synthesis and again following mitosis, while cytoplasmic proteins are excluded. The process is highly specific: a single base change in vertebrate initiator tRNAMet (tRNAiMet) reduces the rate of export 20-fold; a point mutation within the simian virus 40 (SV40) large-T antigen, converting Lys 128 to Thr or Asn, prevents import. Lys 128 lies within a short 'signal' sequence which, when fused to large non-nuclear proteins, causes their accumulation in nuclei. Regions of other eukaryotic proteins also seem to contain nuclear localization signals, although a single consensus sequence has not emerged. We report here that a synthetic peptide containing 10 residues of large-T antigen sequence serves as a nuclear localization signal when cross-linked to bovine serum albumin (BSA) or immunoglobulin G (IgG) and microinjected in Xenopus oocytes. Substitution of Thr at the position of Lys 128 in this peptide renders it six- to sevenfold less effective. The uptake of peptide-linked BSA is saturable, and the rate is diminished by co-injection of free peptide. These findings are indicative of a receptor-mediated uptake process. With the use of anti-peptide antibodies, a family of proteins is revealed in nuclear but not cytoplasmic extracts of human lymphocytes which contain large-T antigen-like sequences.     

Simian virus 40 replication in adenovirus-transformed human cells antagonizes gene expression

Simian virus 40 (SV40) replicates efficiently in monkey kidney cells. However, we have now found that SV40-based vectors transfected into most human cells replicate poorly, if at all. In contrast, strong SV40 replication is observed in human embryonic kidney (HEK) cells transformed with the adenovirus early region, but not in untransformed HEK cells. Vector replication in adenovirus-transformed cells is dependent on the presence of the SV40 origin of replication and large-T antigen. However, vigorous replication occurs at levels of large-T antigen that are undetectable by immunofluorescence. These data suggest that the adenovirus oncogenes create a replication-permissive environment to which the SV40 replicon responds. Furthermore, replication and gene expression seem to be antagonistic on our vectors. High levels of large-T antigen are observed only when vector replication is blocked by mutations in the gene for large-T antigen or the origin of replication, or by direct inhibition of DNA polymerase with aphidicolin.     

Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes

Following the transfer into fertilized mouse eggs of recombinant genes composed of the upstream region of the rat insulin II gene linked to sequences coding for the large-T antigen of simian virus 40, large-T antigen is detected exclusively in the beta-cells of the endocrine pancreas of transgenic mice. The alpha- and delta-cells normally found in the islets of Langerhans are rare and disordered. Well-vascularized beta-cell tumours arise in mice harbouring and inheriting these hybrid oncogenes.     

A common function for polyoma virus large-T and papillomavirus E1 proteins?

Nucleotide sequencing has revealed a common genetic organization for three papillomaviruses: BPV-1 (bovine papillomavirus type 1), HPV-1 (human papillomavirus type 1a) and HPV-6 (human papillomavirus type 6b). Several open reading frames, corresponding to as yet uncharacterized proteins, were observed in these genomes in the region that is required for oncogenic transformation by BPV-1 and for plasmidial maintenance of its genome. The longest of these frames, E1, is also the most conserved between the three viruses; we have compared the amino acid sequence of its putative product ('E1 protein') with those of the large-T proteins of three polyoma viruses and report here significant homologies in their carboxy-terminal halves, extending for over 200 amino acids. Moreover, similar secondary structures were predicted in this region, especially in two blocks of homologous residues, which correspond in the large-T proteins of polyoma and simian virus 40 (SV40) viruses to sites involved in the ATPase and nucleotide-binding activities. These observations suggest that the papillomavirus E1 proteins might have a function in common with the polyoma virus large-T proteins (which are required for the initiation of viral DNA replication). As it was suggested recently that the E1 gene product is involved in maintaining the BPV-1 genome as a plasmid in transformed cells, we speculate that the structural features conserved in these otherwise very different viruses are general characteristics of eukaryotic proteins involved in the control of DNA replication.     

Creation of human tumour cells with defined genetic elements.

During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.     

SV40 enhancer and large-T antigen are instrumental in development of choroid plexus tumours in transgenic mice

We have shown recently that choroid plexus tumours frequently develop in transgenic mice which have developed from fertilized eggs injected with DNA molecules containing both simian virus 40 (SV40) early-region genes and metallothionein (MT) fusion genes, and several lines of mice have now been established in which all of the offspring that inherit the foreign DNA succumb to these tumours at 3-5 months of age (ref. 1 and our unpublished data). Several other tissues, notably thymus and kidney, occasionally also show pathological changes. SV40 large-T antigen protein and messenger RNA are always present in affected tissues at much greater concentrations than in unaffected tissues, suggesting that SV40 early-region genes are preferentially activated in choroid plexus, thymus and kidney and that this activation frequently leads to tumorigenesis in the choroid plexus. To determine which regions of the original constructs are important for this tumorigenesis, we have now tested several derivatives and report here that the large-T antigen is sufficient, that the MT fusion gene is dispensable and that the SV40 enhancer (72-base-pair repeat region) has an important role in directing tumours to the choroid plexus. Deletion of the SV40 enhancer region alone commonly leads to peripheral neuropathy, as well as liver and pancreatic tumours, which are the subject of the accompanying paper. Evidence is presented that these pathologies may result from an enhancing effect of the MT sequences on large-T antigen genes, made possible by removal of the otherwise dominant SV40 enhancer.     

Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes

Transfection of embryo fibroblasts by a human ras oncogene does not convert them into tumour cells unless the fibroblasts are established and immortalized before transfection. The embryo fibroblasts become tumorigenic if a second oncogene such as a viral or cellular myc gene or the gene for the polyoma large-T antigen is introduced together with the ras gene.     

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.     

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.     

Specific interaction between the p53 cellular tumour antigen and major heat shock proteins

The protein p53 is capable of participating in neoplastic transformation and can form specific complexes with the large-T antigen of simian virus 40 (SV40). This interaction probably results in the stabilization of p53 (refs 7,8) and may contribute to SV40-mediated transformation. Several non-SV40-transformed cells also exhibit a stabilized p53 which is present in elevated levels. Recently, this stabilization was shown to coincide with the ability to precipitate a polypeptide (p68) of relative molecular mass (Mr) 68,000-70,000 by anti-p53 monoclonal antibodies. We now report that this co-precipitation indeed represents a specific complex between the two proteins; the complex sediments on a sucrose gradient as a relatively broad peak of 10-14S and can be dissociated in vitro. Furthermore, p68 is the HSP70 heat shock protein cognate, found in elevated levels in a p53-overproducing cell line. On heat-shock treatment of such overproducers, p53 also forms a complex with the related highly inducible HSP68.     

Tumour prevention and rejection with recombinant vaccinia

Tumour-specific antigens (TSA; ref. 1) have been exploited in the diagnosis and imaging of human cancer and anti-TSA antibodies have therapeutic potential. Vaccination with TSA or anti-idiotypic (TSA) antibodies has also been used to control tumour growth in model systems. An effective immune response nevertheless demands copresentation of antigen with host histocompatibility determinants. We therefore examined whether live vaccinia virus recombinants expressing TSA in cells of the vaccinated host might better elicit tumour immunity. Polyoma virus (PY) is tumorigenic in rodents; because killed PY-transformed cells can elicit tumour immunity, a PY-specific TSA has been postulated. Tumorigenesis involves expression of three early PY proteins, large-T (LT), middle-T (MT) and small-T (ST), but their role as TSAs is unclear. We therefore expressed the three T proteins in separate vaccinia recombinants. Rejection of PY tumours was observed in rats immunized with recombinants expressing either LT or MT. Further, tumour-bearing animals could be induced to reject their tumours by inoculation of recombinants.     

The cellular oncogene p53 can be activated by mutagenesis

P53 is a cellular phosphoprotein of short half-life (t1/2) which is present at elevated levels in cells transformed by various stimuli including viruses, chemicals and radiation. p53 forms specific stable complexes with simian virus 40 (SV40) large-T antigen and an adenovirus E1b protein of relative molecular mass (Mr) 57,000. A number of reports have associated p53 with cell proliferation, and p53 complementary DNA expression constructs immortalize primary cells in vitro and render them sensitive to transformation by an activated ras oncogene. We have examined the biological properties of a set of p53 expression constructs, and report here that cellular immortalization by a wild-type p53 cDNA gene is conditional upon the promoter/enhancer construction used, but that p53 can extend cellular lifespan by a second distinct mechanism involving rearrangements of the coding sequence which give rise to stable protein products. Cells immortalized by one of these mutants are refractory to subsequent transformation by a ras oncogene, indicating that cellular immortalization and ras cooperation are separate activities.     

128层螺旋CT三维重建在外伤骨折成像的临床应用

目的研究128层螺旋CT三维重建在外伤骨折成像中的临床应用.方法对36例外伤患者进行128层螺旋CT扫描及三维重建.结果 128层螺旋CT对各个部位的骨折均能良好显示.结论 128层螺旋CT三维重建不仅精确地显示病变的立体形态,还能详细了解各解削结构的空间关系,是目前诊断外伤骨折的理想方法.     

Focus formation in rat fibroblasts exposed to a tumour promoter after transfer of polyoma plt and myc oncogenes

The gene encoding the large-T protein of polyoma virus (plt), the E1A genes of adenoviruses, the viral myc gene (v-myc) or rearranged forms of the cellular c-myc gene confer on rat embryo fibroblast (REF) cells in primary culture a series of new properties ('immortality', reduced serum requirement and sensitivity to transformation by viral and activated cellular oncogenes) but do not induce the appearance of transformed foci. We now report that focus formation can be induced after transfer of these genes into either REF or established FR3T3 rat cells by subsequent exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Frequencies of transformation are in the same range as those usually observed for transformation with complete polyoma DNA or with a mixture of cloned myc and ras oncogenes. These results further characterize the 'immortalized' state induced by plt and myc as one in which the cells maintain a normal growth control in many respects but can be further acted upon to produce a neoplastic progeny.     

黑白花奶牛“白细胞粘附缺陷症”（LAD）的分子生物学分析与诊断

从一患白细胞粘附缺陷症（ＬＡＤ）的黑白花奶牛犊的核苷酸顺序分析中，发现牛的抗原分子分化群１８（ＣＤ１８）的基因密码有二处发生了点突变；一处在第３８３核苷酸上，在此处原有的腺瞟吟已被鸟嘌呤取代；另一处在第７７５核苷酸上，在此处原有的胞嘧啶被胸腺嘧啶取代了。在第７７５核苷酸的突变是静止的，因为它并没有使其相应的氨基酸顺序发生改变。在第３８３核苷酸的突变，使其在相应的氨基酸顺序中第１２８位氨基酸位置上的天门冬氨酸被甘氨酸（Ｄ１２８Ｇ）所替代。一种用于牛的Ｄ１２８Ｇ等位基因的快速筛选诊断方法已作了说明。在美国，在黑白花奶牛中Ｄ１２８Ｇ等位基因的携带率在公牛中占１４．１％，在母牛中占５．８％。这一突变基因已流行于世界各地的黑白花奶牛之中。     

Mouse p53 inhibits SV40 origin-dependent DNA replication

p53 is a cellular phosphoprotein that is present at elevated concentrations in cells transformed by different agents. p53 complementary DNA expression-constructs immortalize primary cells in vitro and co-operate with an activated ras oncogene in malignant transformation. Several reports have implicated p53 in mammalian cell cycle control and specifically with events occurring at the G0-G1 boundary. p53 forms specific complexes with simian virus 40 (SV40) large-T antigen, and such complexes are found associated with both replicating and mature SV40 DNA in lytically infected cells. In an accompanying paper Gannon and Lane report that in in vitro plate-binding assays, mouse p53 can displace polymerase alpha from complex with T-antigen. We have examined the in vivo consequences of expressing wild-type and mutant p53 proteins from other species in SV40-transformed monkey cells. We report here that expression of mouse p53 results in a substantial and selective inhibition of SV40 origin-dependent DNA replication. In addition to any function in the G0-G1 transition, the data presented suggest that p53 may affect directly the initiation or maintenance of replicative DNA synthesis.     

对10轮AES-128的中间相遇攻击

给出了AES-128相邻两轮的轮密钥之间的一个线性关系。通过将这一关系与Hüseyin Demirci和Ali Aydın Selçuk在2008年提出的一个5轮AES区分器相结合,构造了一个8轮AES区分器。在这个8轮AES区分器的基础上,设计了一个对10轮AES-128的中间相遇攻击方案。这一方案在预计算阶段可以节省相当大的存储空间。     

基于Feistel结构的分组密码算法Eslice

一族安全性较高的分组密码算法Eslice,包含3个版本:Eslice-64-64,分组长度和密钥长度均为64比特;Eslice-64-128,分组长度为64比特,密钥长度为128比特;Eslice-128-128,分组长度和密钥长度均为128比特。Eslice的设计灵感源于LBlock。整体采用Feistel结构,轮函数采用SP结构,所选取的S盒其各项密码性质均达到最优。线性变换仅有循环移位和异或两种操作,且密钥生成算法与加密算法使用相同的S盒。进一步,分析Eslice针对差分、线性、积分等密码分析方法的安全性,利用基于混合整数线性规划(MILP)的搜索模型,得到20轮的最小活跃S盒的个数为41个,比LBlock 20轮的最小活跃S盒的个数少3个,通过活跃S盒的个数估算差分概率和线性偏差,对算法进行安全性评估。结果表明,Eslice算法可以有效的抵抗差分攻击和线性攻击。