Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product

The E7 transforming protein of human papilloma virus-16 binds to the retinoblastoma gene product (pRb) through a nine-amino-acid segment of E7 (21-29). This segment of E7 is homologous to the pRb-binding domains of the simian virus 40 large T and adenovirus E1A transforming proteins. Each of these viral transforming proteins bind to the same region of pRb. To isolate cellular proteins that interact with this viral protein-binding domain on pRb, we used recombinant pRb to screen a human complementary DNA expression library. Two cDNAs were isolated that encode retinoblastoma binding proteins (RBP-1 and RBP-2). We report here that these RBP genes exist in separate loci and produce discrete messenger RNAs. The predicted amino-acid sequence of these genes showed no homology to known proteins, but both RBPs contain the pRb binding motif conserved between E7, large T and E1A14. In vitro expression of the RBP cDNAs yielded proteins that specifically bound to pRb. Recombinant E7 protein, the E7 21-29 peptide and the homologous RBP-1 peptide inhibited RBP-pRb binding. Mutations introduced into the putative pRb-binding segment in RBP-1 impaired its binding activity. These studies indicate that the cellular RBP-1, RBP-2 and viral E7 proteins interact with pRb through similar domains.     

A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus E1A products

SV40 large T antigen contains a small region of amino acid sequence, conserved among the papovaviruses, that shows considerable similarity to conserved domain 2 of the adenovirus E1A oncogene, a domain which plays an important role in the E1A transforming functions. To learn whether the analogous SV40 T antigen sequences could substitute functionally for E1A domain 2, a chimaeric gene was constructed, coding for T antigen amino acid residues 101 to 118 in place of E1A domain 2. The resulting product showed much of the activity of the wild-type E1A products. It induced proliferation of primary BRK cells and cooperated with the ras oncogene to transform these cells fully. In addition, the chimaeric protein coprecipitated two cellular proteins whose specific binding to the E1A products depends on the presence of domain 2. The activity of the chimaeric product suggests that a similar functional unit exists in the transforming proteins of both SV40 and adenovirus, and that these proteins may exert their cell growth regulating effects through similar mechanisms.     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system

The human adenovirus type 5 E1A, a tumor- suppressor gene[1], codes for two major related proteins of 243 amino acids (12S) and 289 amino acids (13S) by al-ternative splicing in two exons[2]. Studies have been shown that E1A can regulate expression of many genes and cell cycle[3]. Both in vitro and in vivo experiments indicated that E1A could induce tumor cells differentia-tion, convert tumor cells into an epithelial phenotype, in-hibit tumor cell growth and metastasis and strongly en-ha…     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system on the human tumor cell growth

Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the sensitivity of chemotherapy and radiotherapy. E1A have the ability to integrate into the host genome, resulting in long-time expression that induces Rb gene inactivation and animal cells immortalization. This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy. Thus, we firstly constructed E1A eucaryotic expression vector (pPIC9/E1A), transformated the pichia pastoris yeast cells (GS115) and screened the high-expressing recombinant strains. The positive yeast strains were cultured in the shake flask, and induced for 3 d. The crude E1A protein was purified using two steps of column chromatography on HiTrap Q and HiTrap SP. The purified E1A protein was identified by SDS-PAGE and Western blot. E1A protein was mostly located at cellular nuclear when Chariot delivered E1A protein into cells. The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase, and significantly inhibited the growth of LN686 tumor cells. The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system of the human tumor cell growth

Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the censitivity of chemotherapy and radiotherapy.E1A have the ability to integrate into the host genome,resulting in long-time expres-sion that induces Rb gene inactivation and animal cells im-mortalization.This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy.Thus,we firstly comstructed E1A eu-caryotic expression vector (pPIC9/E1A),transformated the pichia pastoris yeast cells(GS115) and screened the high-expressing recombinant strains.The positive yeast strains were cultured in the shake flask,and induced for 3d.The crude E1A protein was purified using two steps of col-umu chromatography on HiTrap Q and HiTrap SP.The pu-rified E1A protein was identified by SDS-PAGE and Western blot.E1A protein was mostly located at cellular unclear when Cheriot delivered E1A protein into cells.The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase,and significantly inhibited the growth of LN686 tumor cells.The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.     

Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

The proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.     

Effects of an Rb mutation in the mouse.

The retinoblastoma gene is mutated in several types of human cancer and is the best characterized of the tumour-suppressor genes. A mouse strain has been constructed in which one allele of Rb is disrupted. These heterozygous animals are not predisposed to retinoblastoma, but some display pituitary tumours arising from cells in which the wild-type Rb allele is absent. Embryos homozygous for the mutation die between days 14 and 15 of gestation, exhibiting neuronal cell death and defective erythropoiesis.     

Inactivation of the p53 pathway in retinoblastoma

Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.     

胡萝卜抗冻蛋白基因的克隆及其在E.coli中的表达

对中国的一个胡萝卜品种进行了冷诱导前后幼苗总蛋白组成的比较、分析 ,SDS聚丙烯酰胺凝胶电泳的结果表明两者在相对分子质量 36 0 0 0附近有一条蛋白带的差别。由此推测实验的胡萝卜品种在冷诱导过程中有抗冻蛋白 (AFP)表达。根据相关的研究结果设计引物 ,以胡萝卜幼苗基因组DNA为模板 ,通过PCR扩增得到了长 10 99bp的抗冻蛋白基因。测序结果表明其与GenBank公布的afp序列仅有 3个碱基的差别。将此抗冻蛋白基因克隆进大肠杆菌表达载体pGEX4T1,在IPTG的诱导下表达出了含有AFP的约 6 0 0 0 0的融合蛋白 ,证明该AFP基因在原核表达系统中可以正常表达     

The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity

The human gene (RB) that determines susceptibility to hereditary retinoblastoma has been identified recently by molecular genetic techniques. Previous results indicate that complete inactivation of the RB gene is required for tumour formation. As a 'cancer suppressor' gene, RB thus functions in a manner opposite to that of most other oncogenes. Sequence analysis of RB complementary DNA clones demonstrated a long open reading frame encoding a hypothetical protein with features suggestive of a DNA-binding function. To further substantiate and identify the RB protein, we have prepared rabbit antisera against a trypE-RB fusion protein. The purified anti-RB IgG immunoprecipitates a protein doublet with apparent relative molecular mass (Mr) of 110,000-114,000. The specific protein(s) are present in all cell lines expressing normal RB mRNA, but are not detected in five retinoblastoma cell lines examined. The RB protein can be metabolically labelled with 32P-phosphoric acid, indicating that it is a phosphoprotein. Biochemical fractionation and immunofluorescence studies demonstrate that the majority of the protein is located within the nucleus. Furthermore, the protein can be retained by and eluted from DNA-cellulose columns, suggesting that it is associated with DNA binding activity. Taken together, these results imply that the RB gene product may function in regulating other genes within the cell.     

6个氨基酸小C肽人胰岛素原类似物基因的构建和表达

用片段置换法，从在Ｃ肽两端具有酶切位点的双突人胰岛素原因中，将Ｃ肽基因替换成含Ａｒｇ－Ａｒｇ－Ｇｌｙ－Ｓｅｒ－Ａｒｇ－Ｌｙｓ６个氨基酸小Ｃ肽基因。将这个小Ｃ肽岛素原类似物基因重组到具有Ｔａｃ启动子的质粒中，并与部分牛凝乳酶原基因融合，在Ｅ．ｃｏｌｉ中得到了高效表达。表达的ＢＣ＇Ａ融合蛋白占菌体总蛋白的１６％。表达产物以包含体形式存在，经ＣＮＢｒ裂解及磺酸，再经复性后，具有对胰岛素的放射免疫活性。