Murine leukemia viruses: antigenic studies by quantitative complement fixation

The murine leukemia viruses of Rauscher and Friend, derived from plasnma of infected Balb/c mice, was purified. Their antigenic relationship was studied by quantitative complement-fixation reactions with the virion antigen and homologous antiserums. The complement-fixation curves observed in cross-reactions indicated close antigenic similarity between these two leukemia viruses. Highly purified viral preparations contained detectable amounts of host antigens.     

Transgenic monkeys produced by retroviral gene transfer into mature oocytes

Transgenic rhesus monkeys carrying the green fluorescent protein (GFP) gene were produced by injecting pseudotyped replication-defective retroviral vector into the perivitelline space of 224 mature rhesus oocytes, later fertilized by intracytoplasmic sperm injection. Of the three males born from 20 embryo transfers, one was transgenic when accessible tissues were assayed for transgene DNA and messenger RNA. All tissues that were studied from a fraternal set of twins, miscarried at 73 days, carried the transgene, as confirmed by Southern analyses, and the GFP transgene reporter was detected by both direct and indirect fluorescence imaging.     

Murine leukemia: a virus-induced autoimmune disease?

Thymocytes from mice carrying Moloney murine leukemia virus since birth are cytotoxic for normal syngeneic fibroblasts; they are much less cytotoxic for the same cells infected with this virus. The cytotoxic thymocytes appear to increase in number with age of the carrier mice and are present both during preleukemic and leukemic periods. These results suggest that lymphomas in carrier mice result from a sequence of events initiated by intrathymic destruction of normal cells by virus-infected cells, and culminating in the unrestricted proliferation of autoaggressive clones in the thymic cortex.     

High-level recombinant gene expression in rabbit endothelial cells transduced by retroviral vectors

By virtue of its immediate contact with the circulating blood, the endothelium provides an attractive target for retroviral vector transduction for the purpose of gene therapy. To see whether efficient gene transfer and expression was feasible, rabbit aortic endothelial cells were infected with three Moloney murine leukemia virus-derived retroviral vectors. Two of these vectors carry genes encoding products that are not secreted: N2, containing only the selectable marker gene neoR, and SAX, containing both neoR gene and an SV40-promoted adenosine deaminase (ADA) gene. The third vector, G2N, contains a secretory rat growth hormone (rGH) gene and an SV40-promoted neoR gene. Infection with all three vectors resulted in expression of the respective genes. A high level of human ADA expression was observed in infected endothelial cell populations both before and after selection in G418. G2N-infected rabbit aortic endothelial cells that were grown on a synthetic vascular graft continued to secrete rGH into the culture medium. These studies suggest that endothelial cells may serve as vehicles for the introduction in vivo of functioning recombinant genes.     

Murine leukemia virus: high-frequency activation in vitro by 5-iododeoxyuridine and 5-bromodeoxyuridine

Cells of embryos of the high leukemic mouse strain AKR can be grown in culture as virus-negative cell lines. However, these lines and clonal sublines uniformly have the capacity to initiate synthesis of murine leukemia virus. Exposure of the cells to 5-iododeoxyuridine or 5-bromodeoxyuridine induced synthesis of virus in as high as 0.1 to 0.5 percent of the cells; many of the cells were producing virus as soon as 3 days after initiation of treatment. Induction of virus by these drugs is several orders of magnitude greater than that obtained with any other treatment tested. These studies indicate that the full genome of murine leukemia virus is present in an unexpressed form in all AKR cells and provide a potentially powerful technique for activating leukemia virus genomes in other cell systems.     

Murine leukemia virus: restriction in fused permissive and nonpermissive cells

Cultures of human cells nonpermissive for mouse leukemia virus replication could not be induced to support virus replication by homologous fusion in the presence of Moloney leukemia virus. Human cells were also fused with permissive mouse cells, and the fate of the virus in heterokaryons was determined by a simultaneous autoradiography and fluorescent antibody technique. Heterokaryons containing the full chromosome complement of both cells were likewise nonpermissive for virus synthesis, but hybrids of human and mouse cells, which lacked up to half of the human chromosome complement, were permissive for virus synthesis. The results suggest that human cell genes can direct a repressive control over mouse leukemia virus replication.     

Chromosome abnormality in rat leukemia induced by 7,12-dimethylbenz[a]anthracene

A high percentage of consistent chromosome abnormality, trisomy of the longest telocentric chromosome, was found in leukemias induced in rats of the Long-Evans strain by pulse doses of 7,12-dimethylbenz[a]anthracene. Cells with this abnormality were large, immature, and mononuclear and tended toward erythroblastic maturation.     

Neoplastic transformation of rat thymic cells induced in vitro by Gross leukemia virus

Cultures of embryonal rat thymus infected initially with Gross leukemia virus have, at the present time, abundantly replicated infectious virus particles for 20 months. Cells from these cultures, after 3 months in vitro, displayed morphological changes and induced formation of tumors upon isotransplantation. The tumors were serially transplantable, and subsequent transplants continue to carry the initial Gross leukemia virus.     

T-lymphocyte priming and protection against Friend leukemia by vaccinia-retrovirus env gene recombinant

The current prevalence of the acquired immune deficiency syndrome in humans has provoked renewed interest in methods of protective immunization against retrovirus-induced diseases. In this study, a vaccinia-retrovirus recombinant vector was constructed to study mechanisms of immune protection against Friend virus leukemia in mice. The envelope (env) gene from Friend murine leukemia virus (F-MuLV) was inserted into the genome of a vaccinia virus expression vector. Infected cells synthesized gp85, the glycosylated primary product of the env gene. Processing to gp70 and p15E, and cell surface localization, were similar to that occurring in cells infected with F-MuLV. Mice inoculated with live recombinant vaccinia virus had an envelope-specific T-cell proliferative response and, after challenge with Friend virus complex, developed neutralizing antibody and cytotoxic T cells (CTL) and were protected against leukemia. In contrast, unimmunized and control groups developed a delayed neutralizing antibody response, but no detectable CTL, and succumbed to leukemia. Genes of the major histocompatibility complex influenced protection induced by the vaccinia recombinant but not that induced by attenuated N-tropic Friend virus.     

Rapid switching of plant gene expression induced by fungal elicitor

The pattern of messenger RNA synthesis in suspension-cultured bean cells (Phaseolus vulgaris L.) was analyzed by blot hybridization and in vitro translation of newly synthesized messenger RNA. The RNA was separated from preexisting RNA by organomercurial affinity chromatography after in vivo labeling with 4-thiouridine. The elicitor induced the synthesis of messenger RNA's encoding phenylalanine ammonia-lyase, chalcone synthase, and chalcone isomerase, three enzymes of phenylpropanoid metabolism involved in the synthesis of isoflavonoidderived phytoalexins. This is part of a rapid and extensive change in the pattern of messenger RNA synthesis directing production of a set of proteins associated with expression of disease resistance.     

携带人血清白蛋白cDNA的逆转录病毒质粒载体的构建

采用粘平端定向基因克隆技术,成功地构建了携带ＨＳＡｃＤＮＡ基因片段的重组逆转录病毒质粒载体ＧＩＮＨＳＡａ。经限制性酶切鉴定和菌落原位杂交证明ＨＳＡｃＤＮＡ已克隆到Ｇ１Ｎａ当中并得到９个重组病毒质粒载体阳性克隆。     

Construction and recovery of viable retroviral genomes carrying a bacterial suppressor transfer RNA gene

The integration of retroviral genomes into cellular DNA can induce mutations by altering the expression of nearby cellular genes and can serve to identify the gene affected. The construction of a retrovirus that stably carries a suppressor transfer RNA gene from Escherichia coli has allowed facile recovery of the viral genome in vectors marked with amber mutations. This virus can be used for rapid isolation of cellular sequences at the site of proviral insertion.     

光诱导c-fos基因在母鸡中脑的表达

为了解光照对母鸡生产性能的影响,采用c-fos法探讨光信息在母鸡中脑的传导通路,将鸡右眼遮光7 d后接受20 lx的光照刺激1.5 h,暗适应1.5 h后灌流固定,取脑制作石蜡切片,采用免疫组化方法检测c-fos基因在中脑的表达。结果显示:中脑内有大量Fos样免疫反应阳性神经元,主要见于左侧,分布于中脑SGC层、峡核(大细胞部Imc、小细胞部Ipc)、峡视核(ION)、中脑外侧核背侧部(MLd)、红核(RN)、滑车神经核(nIV)。表明光照信息主要通过视网膜经离顶盖通路传达至母鸡中脑,同时顶盖-峡核回路也起重要作用。     

Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer

The 11-kD protease (PR) encoded by the human immunodeficiency virus 1 (HIV-1) is essential for the correct processing of viral polyproteins and the maturation of infectious virus, and is therefore a target for the design of selective acquired immunodeficiency syndrome (AIDS) therapeutics. To facilitate the identification of novel inhibitors of HIV-1 PR, as well as to permit detailed studies on the enzymology and inhibition of this enzyme, a continuous assay for its activity was developed that was based on intramolecular fluorescence resonance energy transfer (RET). The assay used the quenched fluorogenic substrate 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)--Ser Gln Asn Tyr Pro Ile Val Gln--5-[(2-aminoethyl)amino]naphthalene-1 sulfonic acid (EDANS), whose peptide sequence is derived from a natural processing site for HIV-1 PR. Incubation of recombinant HIV-1 PR with the fluorogenic substrate resulted in specific cleavage at the Tyr-Pro bond and a time-dependent increase in fluorescence intensity that was linearly related to the extent of substrate hydrolysis. An internally quenched fluorogenic substrate was also designed that was selectively cleaved by the related PR from avian myeloblastosis virus (AMV). The fluorescence quantum yields of the HIV-1 PR and AMV PR substrates in the RET assay increased by 40.0- and 34.4-fold, respectively, per mole of substrate cleaved. Because of its simplicity, rapidity, and precision in the determination of reaction rates required for kinetic analysis, this method offers many advantages over the commonly used high-performance liquid chromatography- or electrophoresis-based assays for peptide substrate hydrolysis by retroviral PRs.     

河鲀毒素诱导菊黄东方鲀膜转运蛋白基因的表达

为探究参与河鲀肝脏和皮肤累积河鲀毒素(Tetrodotoxin, TTX)的过程及相关调控基因,按每克体质量河鲀0.25μg TTX剂量尾部肌肉注射人工繁育的菊黄东方鲀(Takifugu flavidus),发现肝脏中TTX含量在注射后4 h时最高(19.12 ng/g),24 h下降到8.92 ng/g, TTX主要在肝实质细胞中累积;皮肤中TTX含量一直处于上升趋势,4 h为13.70 ng/g, 24 h为22.91 ng/g; TTX主要在皮肤表皮层、基底层细胞中累积。比较对照组和注射24 h后肝脏和皮肤转录组发现：TTX注射导致肝脏组织338个基因上调、350个基因下调;而皮肤组织有598个基因上调,同时有595个基因下调;RT-qPCR验证slc2a12、slc5a7、slc25a22、slc25a15、slc35a3、arap1、ugt2a2、ggt5、psbp2等膜转运蛋白相关基因在注射TTX后在肝脏和皮肤中的表达水平显著上调,可能与TTX累积过程有关。     

6-BA诱导黄瓜CsaIAAs基因的表达研究

Aux/IAA基因是一类典型的能被生长素诱导表达的基因家族,可以被生长素、细胞分裂素、盐及干旱胁迫等诱导表达。采用Trizol法提取黄瓜幼苗的根、茎、叶、花及卷须的总RNA,经反转录成cDNA,再利用CsaIAAs基因特异性引物进行半定量RT-PCR扩增,琼脂糖凝胶电泳检测CsaIAAs基因的表达情况。结果表明,黄瓜29个CsaIAAs基因中,除了CsaIAA25、CsaIAA26几乎在所有器官中都不表达外,CsaIAA07、CsaIAA09、CsaIAA16和CsaIAA20只在个别器官中表达,如CsaIAAs20只在茎中弱表达,CsaIAA29只在卷须中有弱的表达。而绝大部分的CsaIAAs基因（CsaIAA02、CsaIAA03、CsaIAA04、CsaIAA05、CsaIAA06、CsaIAA08、CsaIAA10、CsaIAA11、CsaIAA13、CsaIAA15、CsaIAA16和CsaIAA19等）在大多数器官中都可以被6-BA诱导表达,尤其是以根、茎、叶和卷须中表达量高。探讨CsaIAAs基因在黄瓜根、茎、叶、花及卷须中的表达情况,可为黄瓜CsaIAAs基因的功能研究提供重要的理论参考。     

Frequent activation of c-kis as a transforming gene in fibrosarcomas induced by methylcholanthrene

The DNA's from two of four methylcholanthrene-induced mouse fibrosarcomas contained transforming genes that were identical in their pattern of restriction endonuclease resistance to inactivation of biologic activity. This transforming gene was identified as the activated homolog of the Kirsten murine sarcoma virus onc gene, v-kis. The finding that a defined carcinogen reproducibly leads to activation of kis as a transforming gene should be of value in elucidating the role of oncogenes in the neoplastic process.