实验基础

The experimental introduction of foreign DNA into the germ line of a mouse is a powerful tool used to identify genetic elements required for appropriate tissue expression, to determine the developmental specificity of gene expression and to determine the consequences of inappropria ...

The human polyomavirus, JCV, is the etiological agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy, or PML. Seroepidemiological studies have indicated that greater than 70% of the human population worldwide is ...

BK virus (BKV), one of two human polyomaviruses, was first isolated in cell culture from the urine of an immunocompromised renal transplant patient in England in 1971 (1). Seroprevalence studies have shown that BK infection is widespread in children throughout the world, and the virus thereaf ...

The Simian virus 40 (SV40) DNA replication system has served as a useful model for studies of DNA synthesis in eukaryotes . One major advantage of this system is that, with the exception of one viral protein, termed T-antigen (T-ag), all of the proteins required for DNA replication are supplied by cells th ...

General properties of human polyomavirus BK (BKV) such as its clinical significance, propagation, and serological assays are described in Chapter 3. In this chapter, two molecular methods for detection of a subtype-specific sequence of BKV are described. Both methods were developed ba ...

The development of soluble cell-free systems that accurately transcribe genes has provided a means to investigate the mechanisms of eukaryotic transcription. The results of many studies have shown that specific DNA cis-acting elements, together with a complex array of protein facto ...

The 5243-bp genome of SV40 contains two transcriptional units (see Fig. 1); the early one, which is first expressed early in the lytic cycle of infection, and the late unit, which is expressed at a significant level only after the onset of viral DNA replication (1). The early genes encode the viral regulato ...

The field of gene therapy has evolved from an investigative curiosity to a major focus of medical research. To date, however, its clinical successes have been few. There are many reasons for this lack of success in clinical studies, but among the most important of these is the inadequacy of gene-delive ...

The use of insect cells and lytic baculovirus for expression of biologically active mammalian proteins has been the method of choice of many investigators. Although prokaryotic expression systems provide higher yields and are technically simpler to use, obtaining biologically ac ...

Many events in the viral life cycle involve protein binding to defined sequences on the viral chromosome. Chromatin immunoprecipitation allows the detection of the in vivo interaction of specific proteins with specific genomic regions. In this technique, living cells are treated with ...

We describe a systematic, high-throughput approach to identify proteins involved in functional adenovirus (Ad)host interactions in vitro and in vivo. We were particularly interested in identifying cellular proteins that interact with fiber knob, which is the moiety within the Ad ca ...

Microinjection of mammalian cells provides a powerful method for analyzing in vivo functions of viral genes and viral gene products. By microinjection, a controlled amount (ranging from several to many thousands of copies) of a viral or cellular gene, a protein product of a gene, a polypeptide f ...

The last 50 yr of molecular biological investigations into human adenoviruses (Ads) have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of the Ad productive infection cycle in permissi ...

Natural-killer (NK) cells are lymphocytes that do not express the CD3 T-cell receptor but do express the CD16 (FcγRIII) and CD56 (isoform of NCAM) in humans or NK1.1 antigen in certain strains of mice. NK cells display spontaneous lytic activity but do not exhibit immunological memory. NK cells are im ...

Natural-killer (NK) cells play an important role in recognizing and eliminating virally infected and transformed cells. To study this process, convenient assays for NK-cell function are required. Conventional NK-cell activity assays measure the release of 51Cr from prelabeled ta ...

This chapter provides a protocol for the large-scale purification of adenovirus type 2 and 5 virions and the soluble major coat protein hexon. The purified virus particles remain intact and are suitable for vector, vaccine, or structural studies and can also be used as seed stock for further rounds ...

All adenoviruses (Ads) sequenced so far encode a single endopeptidase of the cysteine class, named adenain. The Ad2 adenain is a 204-residue, nearly inactive monomer, which is activated during virus maturation by an 11-residue cleavage fragment of capsid protein pVI. This chapter describ ...

This method is designed to measure rates of transcription from adenoviral promoters as a function of the concentrations within infected cells of the promoter(s) of interest. The latter parameter is assessed by quantification of viral DNA by hybridization of membrane-bound DNA followi ...

Entry of the adenovirus (Ad) capsids during the early stages of infection is a multistep process that includes initial attachment of the virus capsid to the cell surface followed by internalization of the virus into early endosomes. The Ad fiber protein, a complex of three apparently identical ...

Members of the family Adenoviridae have been isolated from a large variety of hosts, including representatives from every major vertebrate class from fish to mammals. The high prevalence, together with the fairly conserved organization of the central part of their genomes, make the adeno ...