Viral hit and run-oncogenesis: Genetic and epigenetic scenarios

It is well documented that viral genomes either inserted into the cellular DNA or coreplicating with it in episomal form can be lost from neoplastic cells. Therefore, “hit and run”-mechanisms have been a topic of longstanding interest in tumor virology. The basic idea is that the transient acquisition of a complete or incomplete viral genome may be sufficient to induce malignant conversion of host cells in vivo, resulting in neoplastic development. After eliciting a heritable change in the gene expression pattern of the host cell (initiation), the genomes of tumor viruses may be completely lost, i.e. in a hit and run-scenario they are not necessary for the maintenance of the malignant state. The expression of viral oncoproteins and RNAs may interfere not only with regulators of cell proliferation, but also with DNA repair mechanisms. DNA recombinogenic activities induced by tumor viruses or activated by other mechanisms may contribute to the secondary loss of viral genomes from neoplastic cells. Viral oncoproteins can also cause epigenetic dysregulation, thereby reprogramming cellular gene expression in a heritable manner. Thus, we expect that epigenetic scenarios of viral hit and run-tumorigenesis may facilitate new, innovative experiments and clinical studies in spite of the fact that the regular presence of a suspected human tumor virus in an early phase of neoplastic development and its subsequent regular loss have not been demonstrated yet. We propose that virus-specific “epigenetic signatures”, i.e. alterations of the host cell epigenome, especially altered DNA methylation patterns, may help to identify viral hit and run-oncogenic events, even after the complete loss of tumor viruses from neoplastic cells.     

西南地区基因重组微生物研究生物安全管理的现状调查及对策研究

目的了解西南地区基因重组病原微生物的安全管理现状，并提出相对应的管理办法与措施。方法采用问卷调查的方式对西南地区内从事基因重组微生物研究的实验室从课题承担情况、实验室生物防护和管理情况、人员培训和教育情况等方面进行了抽样调查，对调查结果进行了分析。结果在各实验室，基因重组技术研究和应用范围广泛；有关生物安全的基础设施有待改善，实验室防护级别不够；生物安全规章制度执行力度还有待加强；人员培训有待规范化、制度化。结论应加强有关法规制度和管理体系的建设，注重对人员的宣传、培训和管理以及加强生物防护设施建设等。     

天蚕素 B-黄体生成素释放激素结合部位重组基因的构建及其抑癌功能的研究

目的构建天蚕素B-黄体生成素释放激素结合部位（CB-LHRH’）重组基因,探讨CB-LHRH’蛋白成为新型抗肿瘤靶向药物的可能性。方法设计并人工合成CB-LHRH’重组基因序列,应用昆虫细胞．杆状病毒表达系统表达目的蛋白并通过蛋白斑点印迹法鉴定,采用二甲氧唑黄（XTT）比色法检测不同剂量的目的蛋白对卵巢上皮性癌细胞株SKOV3（LHRH受体阴性）和子宫内膜癌细胞株HEC-1B（LHRH受体阳性）细胞的生长抑制率,并进行镜下形态观察。结果蛋白斑点印迹法检测结果显示,重组杆状病毒感染的Si9细胞裂解液呈现棕色,证实CB-LHRH’蛋白已表达。重组杆状病毒感染的昆虫细胞系Sf9细胞裂解液对SKOV3及HEC-1B细胞的生长抑制率,随作用时间的延长和剂量的增加而增加,SKOV3细胞的生长抑制率从（5.03±0.08）％增加至（53.24±1.22）％,HEC-1B细胞的生长抑制率从（5.13±0.37）％增加至（56.16±1.08）％。相同剂量的重组杆状病毒感染的St9细胞裂解液,作用相同时间,其对HEC-1B细胞的生长抑制率高于SKOV3细胞,差异有统计学意义（P〈0.01）。经重组杆状病毒感染的St9细胞裂解液作用后,镜下可见癌细胞出现明显改变甚至成片坏死崩解为无定形的颗粒状物质。结论CB-LHRH’蛋白可有效杀伤LHRH受体阳性的HEC-1B细胞,有望成为治疗表达LHRH受体的肿瘤的抗肿瘤靶向药物。     

pcDNA3.1-HPV18E7重组基因的克隆

目的 从HPV18感染细胞中提取HPV18E7 DNA,构建pcDNA3．1-HPV18E7重组质粒。方法 应用PCR方法从HPV18感染细胞中扩增HPV18E7 DNA,并应用基因重组技术构建pcDNA3．1-HPV18E7重组质粒。结果 PCR扩增产物经电泳检测及测序鉴定证实为HPV18E7;基因重组产物经PCR鉴定及测序证实为pcDNA3．1-HPV18E7,成功构建了pcDNA3．1-HPV18E7重组质粒。结论 从受感染人体细胞中成功扩增出HPV18E7,并成功构建pcDNA3．1-HPV18E7重组质粒,为HPV基因疫苗的构建和治疗HPV感染相关肿瘤奠定了基础。     

Mixed vaginal infections of Balb/c mice with low virulent herpes simplex type 1 strains result in restoration of virulence properties: vaginitis/vulvitis and neuroinvasiveness

Vaginal infections of BALB/c Ann mice with herpes simplex virus type 1 (HSV-1) were studied. Mice were inoculated with virulent strains ANG path and 17 syn⁺ or low-virulent recombinant strains 27/III and 17-syn3 that differ from parental strains in their glycoprotein B (gB) gene sequences. When low-virulent strains were inoculated separately, no vaginitis/vulvitis was produced despite replication in the vagina. In contrast, after coinfection of mice with the two low-virulent strains, vaginitis/vulvitis was produced and virus could be recovered from the central nervous system (CNS). Two of the CNS isolates produced vaginitis/vulvitis, neuroinvasiveness and death of mice after vaginal infection. Restriction fragment analysis and sequencing were used to assess recombination events in the gB gene sequence of the CNS isolates. After mixed vaginal infection recombination between non-virulent HSV strains occurs, resulting in vaginitis/vulvitis and neuroinvasiveness. No correlation was detected between the syncytial phenotype and local vaginal virulence. Virulence of HSV is not solely dependent on gB function; it seems to be more probable that several genes act in concert to induce virulence and neuroivasiveness. Received: 28 May 1996     

人可溶性转化生长因子βⅡ型受体真核表达载体的构建及表达

目的:构建人转化生长因子βⅡ型受体胞外区(sTGFβRⅡ)基因的真核表达载体,转染真核细胞CHO以表达sTGFβRⅡ蛋白,并检测其生物学活性.方法:以TGFβRⅡ的重组质粒为模板,用PCR方法扩增得到人TGFβRⅡ胞外区(1-159位氨基酸)的cDNA,将该cDNA重组到真核表达载体pCDNA3.1/myc-his(-)B(pCDNA)的EcoRⅠ和HindⅢ多克隆位点上,构建成pCDNA-sTGFβRⅡ重组质粒.经双酶切和核酸测序鉴定,将该重组质粒转染CHO细胞,用Western blotting法检测sTGFβRⅡ的表达,并测定其生物学活性.结果:CHO细胞转染pCDNA-sTGFβRⅡ重组质粒后,其培养上清经 Western blotting分析,显示有特异性的蛋白条带,所表达的蛋白能明显抑制TGFβ1对貂肺上皮细胞的增殖抑制作用.结论:成功克隆了sTGFβRⅡ基因,并获得有生物学活性的sTGFβRⅡ蛋白.     

Cloning the REC1 gene of Ustilago maydis

Summary The REC1 gene of Ustilago maydis plays a key role in homologous recombination and the repair of damaged DNA. In order to understand the nature and functions of the gene product, the gene has been cloned by functional complementation. A 3.8 kb cloned fragment complements the pleiotropic mitotic phenotype of different rec1 alleles. It does not complement the UV sensitivity of two other sensitive mutants. Disruption of the chromosomal copy of the 1.566 kb open reading frame within this fragment reproduces the rec1 pleiotropic phenotype. Furthermore, in diploids this disrupted reading frame is unable to complement previously characterised rec1 alleles.     

Superinfection and recombination of infectious laryngotracheitis virus vaccines in the natural host

Infectious laryngotracheitis virus (ILTV, Gallid alphaherpesvirus 1) causes severe respiratory disease in chickens and has a major impact on the poultry industry worldwide. Live attenuated vaccines are widely available and are administered early in the life of commercial birds, often followed by one or more rounds of revaccination, generating conditions that can favour recombination between vaccines. Better understanding of the factors that contribute to the generation of recombinant ILTVs will inform the safer use of live attenuated herpesvirus vaccines. This study aimed to examine the parameters of infection that allow superinfection and may enable the generation of recombinant progeny in the natural host. In this study, 120 specific-pathogen free (SPF) chickens in 8 groups were inoculated with two genetically distinct live-attenuated ILTV vaccine strains with 1–4 days interval between the first and second vaccinations. After inoculation, viral genomes were detected in tracheal swabs in all groups, with lowest copies detected in swabs collected from the groups where the interval between inoculations was 4 days. Superinfection of the host was defined as the detection of the virus that was inoculated last, and this was detected in tracheal swabs from all groups. Virus could be isolated from swabs at a limited number of timepoints, and these further illustrated superinfection of the birds as recombinant viruses were detected among the progeny. This study has demonstrated superinfection at host level and shows recombination events occur under a very broad range of infection conditions. The occurrence of superinfection after unsynchronised infection with multiple viruses, and subsequent genomic recombination, highlight the importance of using only one type of vaccine per flock as the most effective way to limit recombination.     

Sequence types diversity of Legionella pneumophila isolates from environmental water sources in Guangzhou and Jiangmen,China

In this study, 159 Legionella pneumophila strains isolated from various natural and artificial water sources in Guangzhou and Jiangmen, China, were subjected to genotyping by the sequence-based typing (SBT) scheme. These isolates were assigned into 53 sequence types (STs) (50 STs with seven loci data and three unidentified STs with incomplete loci profiles) with ST1 as the dominant one (14.5%), and the index of diversity (IOD) was 0.950. Eight new alleles and 34 new STs were reported here. Notably, most of the newly identified STs with seven loci data (24/34) contained no new allele, implying frequent recombination events in L. pneumophila. Five intragenic recombination events were identified in the concatenated sequences of seven loci. The diversity of STs in natural environmental isolates (41 STs, IOD = 0.956) is higher than that of artificial environmental ones (17 STs, IOD = 0.824). The ST patterns varied in isolates from these two sources: the most common STs from artificial water sources, ST1 and ST752 (39.2% and 13.7%), were only occasionally isolated from natural water sources (2.9% and 3.8%, respectively); while the predominant STs from natural water sources, ST1048, ST739 and ST1267 (15.2%, 6.7% and 6.7%), were less frequently seen in artificial environments (2.0%, 0% and 0%, respectively). We also found out that Legionnaires’ disease associated STs might be more frequently isolated in artificial environments than in natural ones. Our data revealed remarkable genetic diversity of L. pneumophila isolates from environmental water systems of Guangzhou and Jiangmen, and the different ST distribution patterns between natural water and artificial water sources as well.