Oncogenicity of human N-ras oncogene and proto-oncogene introduced into retroviral vectors.

The N-ras gene is the only member of the ras family which has never been naturally transduced into a retrovirus. In order to study the in vitro and in vivo oncogenicity of N-ras and to compare its pathogenicity to that of H-ras, we have inserted an activated or a normal form of human N-ras cDNA into a slightly modified Harvey murine sarcoma virus-derived vector in which the H-ras p21 coding region had been deleted. The resulting constructions were transfected into NIH 3T3 cells. The activated N-ras-containing construct (HSN) induced 10(4) foci per microgram of DNA and was found to be as transforming as H-ras was. After infection of the transfected cells by either the ecotropic Moloney murine leukemia virus or the amphotropic 4070A helper viruses, rescued transforming viruses were injected into newborn mice. Both pseudotypes of HSN virus containing activated N-ras induced the typical Harvey disease with similar latency. However, we found that the virus which contained normal N-ras p21 (HSn) was also pathogenic and induced splenomegaly, lymphadenopathies, and sarcoma in mice after a latency of 3 to 7 weeks. In addition, Moloney murine leukemia virus pseudotypes of N-ras caused neurological disorders in 30% of the infected animals. These results differed markedly from those of previous experiments in which we had inserted the activated form of N-ras in the pSV(X) vector: the resulting SVN-ras virus was transforming on NIH 3T3 cells but was poorly oncogenic in vivo (M. Souyri, C. F. Koehne, P. V. O'Donnel, T. H. Aldrich, M. E. Furth, and E. Fleissner, Virology 158:69-78). However, similarly poor oncogenicity was also observed when the v-H-ras coding sequence was inserted in pSV(X) vector, which indicated that the vector sequences play a crucial role in the pathogenicity of a given oncogene. Altogether, these data demonstrated unequivocally that N-ras is potentially as oncogenic as H-ras and that such oncogenic effect could depend on the vector environment.     

Mechanism of activation of an N-ras gene in the human fibrosarcoma cell line HT1080.

A full length N-ras gene has been cloned from both the human fibrosarcoma cell line HT1080 and from normal human DNA. N-ras isolated from HT1080 will efficiently induce morphological transformation of NIH/3T3 cells in a transfection assay, whereas N-ras isolated from normal human DNA has no effect on NIH/3T3 cells. The coding regions of the normal N-ras gene have been sequenced and the predicted amino acid sequence of the N-ras product is very similar to that of the c-Ha-ras1 and c-Ki-ras2 products. By making chimeric molecules between the two cloned genes the activating alteration in the HT1080 N-ras gene has been localised to a single base change that results in an amino acid alteration at position 61 of the p21 N-ras product.     

Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

Production of chimeric protein coded by the fused viral H-ras and human N-ras genes in Escherichia coli

A new method for the production of a chimeric protein of two related genes has been developed. The nucleotide sequences of the region from the N terminus to the 86th amino acid (aa) residue of human N-ras and of the Harvey sarcoma virus (Ha-MuSV) H-ras are 80% homologous. We isolated the DNA fragment encoding the N-terminal portion up to the 70th aa residue from plasmid pH-1 which encodes the total genome of Ha-MuSV, and the DNA fragment encoding the C-terminal portion from the 40th aa to the C terminus from plasmid p6a1 which includes the human N-ras cDNA but lacks the N-terminal portion. After partial digestion of both fragments with phage lambda exonuclease, which creates 3'-protruding ends, a hybrid was formed between 73% homologous single-stranded DNA portions at the 3' ends of both fragments. The hybrid was recloned on pBR322 after repairing with Escherichia coli DNA polymerase I and DNA ligase. The chimeric v-H/N-ras gene composed of the N-terminal portion of v-H-ras gene and the remaining region of N-ras gene was inserted into an expression vector containing two tandem trp promoters and a terminator, and expressed in E. coli. The chimeric protein was found to accumulate to approx. 10% of total cellular proteins.     

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.     

Concomitant K- and N-ras gene point mutations in clonal murine lymphoma.

We have surveyed a panel of induced murine lymphomas for c-ras gene mutations. The K-ras gene seems to be preferentially activated in our system, and there are at least two examples of concomitant K- and N-ras gene mutations in the same tumor. This indicates that in some cases additional ras mutations may contribute to tumorigenesis and is evidence for a role of ras activation in tumor progression.     

人N-ras癌基因细胞型特异DNA结合蛋白

本文利用Southwestern印迹技术发现人肿瘤HT1080细胞染色质蛋白中一组与N-ras基因结合的蛋白,分子量约为150,105,95,90KDa,而与Ha-ras基因结合的一组蛋白,分子量约为160,115,100,55KDa,其中150KDa蛋白是N-ras基因特异的DNA结合蛋白,具有细胞型特异性,在HT1080细胞中含量最多,T24细胞次之,而在人HeLa细胞,淋巴细胞、肠细胞以及未转化的NTH3T3细胞中未被发现。此种蛋白可能与N-ras基因在HT1080细胞内的激活有密切关系。     

Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs

The murine double minute 2 (mdm2) gene is essential for embryogenesis in mice that express the p53 tumor suppressor protein. Mdm2 levels must be regulated tightly because overexpression of mdm2 contributes to tumorigenesis. We investigated whether the 5' and 3' untranslated regions (UTRs) of murine mdm2 affect the expression of MDM2 proteins. Induction of mdm2 expression by p53 results in synthesis of an mdm2 mRNA with a short 5' UTR. The long 5' UTR increases internal initiation of translation of a minor MDM2 protein, p76(MDM2), without affecting the efficiency of translation of the full-length p90(MDM2). We discovered two alternative 3' untranslated regions in murine mdm2 mRNA expressed in the testis. The longer 3' UTR contains a consensus instability element, but mdm2 mRNAs containing the long and short 3' UTRs have comparable half-lives. The 3' UTRs do not affect either initiation codon use or translation efficiency. Thus, the murine 5' UTR, but not the 3'UTR, influences the ratio of the two MDM2 proteins but neither UTR affects MDM2 abundance significantly.     

Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.     

The product of unr, the highly conserved gene upstream of N-ras, contains multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif.

We show that the open reading frame transcribed from the unr gene (immediately upstream of N-ras) in mammals consists of multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif found in prokaryotic cold-shock proteins, and eukaryotic DNA-binding proteins. Alignment of the CSD sequences of unr with those from other proteins reveals a core of similarity for which a consistent secondary structure prediction can be derived. This prediction suggests that the CSD consists primarily of beta-sheet, in contrast to most known eukaryotic DNA-binding proteins. Sequence analysis of the 3' end of the guinea pig unr gene shows that the core of one CSD repeat is encoded in a single exon, consistent with the modular assembly of the gene from ancestral CSD-coding units.