Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Integrated State of Oncornavirus DNA in Normal Chicken Cells and in Cells Transformed by Avian Myeloblastosis Virus

The covalent linkage of oncornavirus-specific DNA to chicken DNA was investigated in normal chicken embryo fibroblasts (CEF) and in virus-producing leukemic cells transformed by avian myeloblastosis virus (AMV). The virus-specific sequences present in cellular DNA fractionated by different methods were detected by DNA-RNA hybridization by using 70S AMV RNA as a probe. In CEF and in leukemic cells, the viral DNA appeared to be present only in the nucleus. After cesium chloride-ethidium bromide density equilibrium sedimentation, the viral DNA was present as linear, double-stranded molecules not separable from linear chicken DNA. After extraction by the Hirt procedure, the viral DNA precipitated with the high-molecular-weight DNA. After alkaline sucrose velocity sedimentation, the viral DNA cosedimented with the high-molecular-weight cellular DNA. The results indicate that in both types of cells studied, the oncornavirus-specific DNA sequences were linked by alkali stable bonds to nuclear cellular DNA of high molecular weight and did not appear to be present in free form of any size.     

Expression of the Major Internal Viral Polypeptide in Cells Transformed by Wild-Type and Temperature-Sensitive Murine Sarcoma Virus

Phenotypic expression of the murine intraspecies and interspecies antigenic determinants of the major type C viral structural 30,000-dalton polypeptide, p30, was measured by radioimmunoassay inhibition in cell lines from different species. Uninfected normal rat kidney (NRK) cells did not contain detectable levels of murine intraspecies and interspecies p30 antigen, whereas rat cells transformed by and producing murine sarcoma virus (MSV)-Moloney leukemia virus (M-MSV-MuLV) contained high levels of both murine intraspecies and interspecies p30 antigen. Significant amounts of murine intraspecies and interspecies p30 antigen were detected in wild-type MSV-transformed nonproducer NRK cells. The control of p30 antigen expression was examined in temperature-sensitive MSV-transformed nonproducer cells [NRK(MSV-1b)] which are cold sensitive for maintenance of the transformed phenotype. Both murine intraspecies and interspecies p30 antigens were detected in NRK(MSV-1b) cells when grown at the permissive (39 C) or nonpermissive (33 C) temperature, suggesting that p30 antigen expression is not correlated with maintenance of the transformed phenotype. The results demonstrate that previously undetectable p30 antigens are expressed in MSV-transformed nonproducer NRK cells, and suggest that the expression of p30 antigen may be a useful marker for viral gene expression in mammalian cells.     

Detection of the Viral Sarcoma Gene Product in Cells Infected with Various Strains of Avian Sarcoma Virus and of a Related Protein in Uninfected Chicken Cells

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.     

Use of DNA-DNA Annealing to Detect New Virus-Specific DNA Sequences in Chicken Embryo Fibroblasts After Infection by Avian Sarcoma Virus

Labeled, virus-specific DNA synthesized in vitro by the virion-associated polymerase of avian sarcoma virus (ASV) was used to measure virus-specific sequences in cell DNA in three ways: (i) by determining the effect of cell DNA upon the reassociation rate of double-stranded polymerase products; (ii) by measuring the kinetics of annealing of single-stranded polymerase product (cDNA) to cell DNA; or (iii) by measuring the amount of cDNA which anneals to a large excess of cell DNA. With these three assays and modifications of them, we show that fewer than five copies of ASV-specific DNA sequences are present per diploid cell in uninfected chicken embryos; that a two- to several-fold increase in copy number of viral DNA follows infection by ASV; that infection introduces to the cell viral sequences not present before infection; and that DNAs from uninfected Pekin duck and Japanese quail embryos show no homology with DNA synthesized by the ASV polymerase. Some of these results differ from data in a previous report from this laboratory (H. E. Varmus, R. A. Weiss, R. R. Friis, W. Levinson, and J. M. Bishop, 1972) and, in general, reconcile our observations with those from other laboratories.     

Virus Deoxyribonucleic Acid Sequences in Subdiploid and Subtetraploid Revertants of Polyoma- Transformed Cells

Polyoma-transformed cells can revert in the properties characteristic of transformation, although they maintain the polyoma-specific T antigen. Transformed cells contain the same number of copies of polyoma virus deoxyribonucleic acid (DNA) per cell (eight) as revertants with a subdiploid or a subtetraploid chromosome number. The results indicate that the duplication of chromosomes in the subtetraploid revertants did not include the chromosomes that carry the viral genome. The virus DNA in both transformed and revertant cells was associated with high-molecular-weight cell DNA. Reversion of the properties of transformed cells was, therefore, not associated either with a decrease in number of virus DNA copies per cell or with a lack of association of the virus DNA with cell DNA.     

Replication of Radiation-Induced Murine Leukemia Virus in Normal and Transformed Mouse Cells

Autonomous radiation-induced leukemia virus (RadLV) replication could be detected in mouse 3T3 cells by the development of interference with murine sarcoma virus (MSV), the appearance of covert helper activity for defective MSV, and by the induction of cytopathic effect type foci in MSV-transformed, leukemia virus-negative (S+L-) cells. A chronic infection of either 3T3 or S+L- cells with RadLV could be established. Both RadLV infectivity and helper activity were demonstrated in the same peak at a buoyant density of 1.16 g/cm(3). Additionally a soluble inhibitor of MSV focus formation was found which could be separated from infectious RadLV. Examination of cell clones derived from chronically infected 3T3 cells showed that essentially every cell was infected and produced both infectious RadLV and low levels of inhibitor. Quantitative comparisons of autonomously replicating RadLV in normal 3T3 and S+L- cells suggested that RadLV may consist of several populations of virus of varying replicative potential. Apparently 99% of RadLV can be assayed only as helper units in normal cells or as replicative units in S+L- cells. To explain the atypical results, a model for RadLV deficiency is proposed.     

DNA and RNA from Uninfected Vertebrate Cells Contain Nucleotide Sequences Related to the Putative Transforming Gene of Avian Myelocytomatosis Virus

The avian carcinoma virus MC29 (MC29V) contains a sequence of approximately 1,500 nucleotides which may represent a gene responsible for tumorigenesis by MC29V. We present evidence that MC29V has acquired this nucleotide sequence from the DNA of its host. The host sequence which has been incorporated by MC29V is transcribed into RNA in uninfected chicken cells and thus probably encodes a cellular gene. We have prepared radioactive DNA complementary to the putative MC29V transforming gene (cDNA(mc) (29)) and have found that sequences homologous to cDNA(mc) (29) are present in the genomes of several uninfected vertebrate species. The DNA of chicken, the natural host for MC29V, contains at least 90% of the sequences represented by cDNA(mc) (29). DNAs from other animals show significant but decreasing amounts of complementarity to cDNA(mc) (29) in accordance with their evolutionary divergence from chickens; the thermal stabilities of duplexes formed between cDNA(mc) (29) and avian DNAs also reflect phylogenetic divergence. Sequences complementary to cDNA(mc) (29) are transcribed into approximately 10 copies per cell of polyadenylated RNA in uninfected chicken fibroblasts. Thus, the vertebrate homolog of cDNA(mc) (29) may be a gene which has been conserved throughout vertebrate evolution and which served as a progenitor for the putative transforming gene of MC29V. Recent experiments suggest that the putative transforming gene of avian erythroblastosis virus, like that of MC29V, may have arisen by incorporation of a host gene (Stehelin et al., personal communication). These findings for avian erythroblastosis virus and MC29V closely parallel previous results, suggesting a host origin for src (D. H. Spector, B. Baker, H. E. Varmus, and J. M. Bishop, Cell 13:381-386, 1978; D. H. Spector, K. Smith, T. Padgett, P. McCombe, D. Roulland-Dussoix, C. Moscovici, H. E. Varmus, and J. M. Bishop, Cell 13:371-379, 1978; D. H. Spector, H. E. Varmus, and J. M. Bishop, Proc. Natl. Acad. Sci. U.S.A. 75:4102-4106, 1978; D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature [London] 260:170-173, 1976), the gene responsible for tumorigenesis by avian sarcoma virus. Avian sarcoma virus, avian erythroblastosis virus, and MC29V, however, induce distinctly different spectra of tumors within their host. The putative transforming genes of these viruses share no detectable homology, although sequences homologous to all three types of putative transforming genes occur and are highly conserved in the genomes of several vertebrate species. These data suggest that evolution of oncogenic retroviruses has frequently involved a mechanism whereby incorporation and perhaps modification of different host genes provides each virus with the ability to induce its characteristic tumors.     

Presence of Cell and Virus Specific Sequences in the Same Molecules of Nuclear RNA from Virus Transformed Cells

Hybridization studies show that nuclear RNA in transformed cells contains both host and virus specific sequences. This suggests virus-specific mRNA could be formed by selective cleavage of high molecular weight nuclear RNA.     

Integration of New Genes into Cellular Networks,and Their Structural Maturation

It has been recently discovered that new genes can originate de novo from noncoding DNA, and several biological traits including expression or sequence composition form a continuum from noncoding sequences to conserved genes. In this article, using yeast genes I test whether the integration of new genes into cellular networks and their structural maturation shows such a continuum by analyzing their changes with gene age. I show that 1) The number of regulatory, protein–protein, and genetic interactions increases continuously with gene age, although with very different rates. New regulatory interactions emerge rapidly within a few million years, while the number of protein–protein and genetic interactions increases slowly, with a rate of 2–2.25 × 10⁻⁸/year and 4.8 × 10⁻⁸/year, respectively. 2) Gene essentiality evolves relatively quickly: the youngest essential genes appear in proto-genes ∼14 MY old. 3) In contrast to interactions, the secondary structure of proteins and their robustness to mutations indicate that new genes face a bottleneck in their evolution: proto-genes are characterized by high β-strand content, high aggregation propensity, and low robustness against mutations, while conserved genes are characterized by lower strand content and higher stability, most likely due to the higher probability of gene loss among young genes and accumulation of neutral mutations.     

辣椒抗烟草花叶病毒相关基因研究进展

病毒病是危害辣椒生产的主要病害之一。烟草花叶病毒(TMV)是最早被发现的病毒,它引起的烟草花叶病毒病是多种作物的重要病害,给辣椒等茄科作物的生产带来重大损失。文中综述了辣椒抗TMV防御反应中的相关基因及其研究进展,为明确辣椒抗TMV机理,挖掘抗病基因,选育抗病材料提供参考。     

Morphogenesis of Avian Infectious Bronchitis Virus and a Related Human Virus (Strain 229E)

Avian infectious bronchitis virus (IBV) and strain 229E, a virus recently recovered from patients with colds, have been shown to possess a similar distinctive morphology in negatively stained preparations. An electron microscopic study of the morphogenesis of IBV in the chorioallantoic membrane and of strain 229E in WI-38 cells was performed. In infected cells, round electron-dense particles 82 mmu in diameter were observed to form by a process of budding from membranes of the endoplasmic reticulum and cytoplasmic vesicles. The particles in IBV-infected cells were similar in size and shape to those in strain 229E-infected cells but showed certain differences in internal structure. The evidence that the particles represent virions and the implications of these findings in the classification of this virus group are discussed.     

Expression of a New Complement-fixing Antigen reactive with Murine Sarcoma Virus Rat Antiserum in Rat Cells transformed by Polyoma Virus

WE reported accelerated transformation by DNA viruses (SV40 and polyoma) in rat embryo (RE) cells chronically infected with a C-type RNA virus^1,2. Recently we found in RE cells transformed by polyoma virus a new complement-fixing (CF) antigen detectable by rat antisera having broad reactivity with the various intraspecies and interspecies antigens of the RNA tumour viruses^3–8; this antigen, however, was distinct from the murine intraspecies and interspecies group-specific (gs) antigens both immunologically and by virtue of other properties. It is also distinct from the polyoma virion (capsid) and tumour (“T”) antigens.     

三个苹果NBS基因的鉴定及其对外源生长物质的响应

为了探讨苹果NBS基因的生理作用,从苹果中鉴定了NBS家族的3个基因,分别命名为MdNBS、MdTIR-NBS1和MdTIR-NBS-LRR1。这3个基因编码的蛋白质均含有NB-ARC结构域,MdTIR-NBS1和MdTIR-NBS-LRR1蛋白N端还有TIR结构域,MdTIR-NBS-LRR1蛋白在C端含有LRR结构。荧光定量PCR分析表明,这3个不同类型的NBS基因的表达具有明显不同的组织特异性,此外,MdNBS、MdTIR-NBS1和MdTIR-NBS-LRR1在‘嘎啦’苹果幼苗叶片中均受SA和MeJA诱导。ACC处理不能提高MdNBS和MdTIR-NBS1基因的表达,但能诱导MdTIR-NBS-LRR1基因表达。结果表明,MdNBS、MdTIR-NBS1和MdTIR-NBS-LRR1基因可能参与了苹果抗逆或抗病防御反应。     

人高致病性禽流感病毒H5N1安徽株HA、M2蛋白双顺反子表达载体的构建及表达研究

将我国分离的人H5N1亚型禽流感病毒A/Anhui/1/2005作为研究对象,扩增其HA和M2基因片段并克隆至DNA疫苗表达载体pVRC中,构建成真核表达质粒。为提高HA的表达量,按照人偏爱密码子将HA基因进行优化改造,经全基因合成后插入真核表达载体pVRC,以β-actin蛋白为内参比证明了优化后的HA蛋白表达效果明显提高。将M2基因和优化后的HA基因共同克隆入双顺反子表达载体pIRES中,获得同时表达HA或M2的双顺反子真核表达质粒;通过Western blot和间接免疫荧光检测方法,确认构建的重组质粒在真核细胞中成功地表达了目的蛋白HA和M2。通过上述结果为进一步开展人高致病性禽流感病毒安徽株HA和M2基因的功能与致病性研究及使用表达HA和M2蛋白进行新型人用禽流感双价疫苗研发奠定基础。     

Expression and Replication Studies to Identify New Candidate Genes Involved in Normal Hearing Function

Considerable progress has been made in identifying deafness genes, but still little is known about the genetic basis of normal variation in hearing function. We recently carried out a Genome Wide Association Study (GWAS) of quantitative hearing traits in southern European populations and found several SNPs with suggestive but none with significant association. In the current study, we followed up these SNPs to investigate which of them might show a genuine association with auditory function using alternative approaches. Firstly, we generated a shortlist of 19 genes from the published GWAS results. Secondly, we carried out immunocytochemistry to examine expression of these 19 genes in the mouse inner ear. Twelve of them showed distinctive cochlear expression patterns. Four showed expression restricted to sensory hair cells (Csmd1, Arsg, Slc16a6 and Gabrg3), one only in marginal cells of the stria vascularis (Dclk1) while the others (Ptprd, Grm8, GlyBP, Evi5, Rimbp2, Ank2, Cdh13) in multiple cochlear cell types. In the third step, we tested these 12 genes for replication of association in an independent set of samples from the Caucasus and Central Asia. Nine out of them showed nominally significant association (p<0.05). In particular, 4 were replicated at the same SNP and with the same effect direction while the remaining 5 showed a significant association in a gene-based test. Finally, to look for genotype-phenotype relationship, the audiometric profiles of the three genotypes of the most strongly associated gene variants were analyzed. Seven out of the 9 replicated genes (CDH13, GRM8, ANK2, SLC16A6, ARSG, RIMBP2 and DCLK1) showed an audiometric pattern with differences between different genotypes further supporting their role in hearing function. These data demonstrate the usefulness of this multistep approach in providing new insights into the molecular basis of hearing and may suggest new targets for treatment and prevention of hearing impairment.     

High Basal Expression of Interferon-Stimulated Genes in Human Bronchial Epithelial (BEAS-2B) Cells Contributes to Influenza A Virus Resistance

Respiratory epithelial cells play a key role in influenza A virus (IAV) pathogenesis and host innate response. Transformed human respiratory cell lines are widely used in the study of IAV−host interactions due to their relative convenience, and inherent difficulties in working with primary cells. Transformed cells, however, may have altered susceptibility to virus infection. Proper characterization of different respiratory cell types in their responses to IAV infection is therefore needed to ensure that the cell line chosen will provide results that are of relevance in vivo. We compared replication kinetics of human H1N1 (A/USSR/77) IAVs in normal primary human bronchial epithelial (NHBE) and two commonly used respiratory epithelial cell lines namely BEAS-2B and A549 cells. We found that IAV replication was distinctly poor in BEAS-2B cells in comparison with NHBE, A549 and Madin-Darby canine kidney (MDCK) cells. IAV resistance in BEAS-2B cells was accompanied by an activated antiviral state with high basal expression of interferon (IFN) regulatory factor-7 (IRF-7), stimulator of IFN genes (STING) and IFN stimulated genes (ISGs). Treatment of BEAS-2B cells with a pan-Janus-activated-kinase (JAK) inhibitor decreased IRF-7 and ISG expression and resulted in increased IAV replication. Therefore, the use of highly resistant BEAS-2B cells in IAV infection may not reflect the cytopathogenicity of IAV in human epithelial cells in vivo.