Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation.

The standard chromatin immunoprecipitation (ChIP) assay is used to examine the specific association of transcription factors with DNA in the context of living cells. Here we review two modifications to this protocol which are designed to identify novel target genes of transcription factors in mammalian cells. The main advantage to both of these approaches is that only DNA sequences directly bound by a factor within the context of a living cell will be identified.

Identification and characterization of a novel replicative intermediate of heron hepatitis B virus.

We have identified and characterized a novel intracellular DNA replicative intermediate that is synthesized by heron hepatitis B virus (HHBV) and not by other avian hepadnaviruses. The new DNA form is synthesized in all host cells tested. The HHBV nucleic acid template, and not HHBV proteins, is responsible for the formation of the new form.

Using disulfide bond engineering to study conformational changes in the beta'260-309 coiled-coil region of Escherichia coli RNA polymerase during sigma(70) binding.

RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the beta' subunit (beta'260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with sigma(70) both in vitro and in vivo.

Targeted gene delivery to skin cells in vivo: a comparative study of liposomes and polymers as delivery vehicles.

Liposomes are microscopic lipid membrane vesicles that provide a current strategy for topical, dermal delivery of biologically active molecules. They have been successfully used for the delivery of various low and high molecular weight molecules into the skin, and as an alternative to virus-mediated delivery systems, have opened the field of dermal gene therapy. The present study was undertaken on 6-day-old rat pups to determine in vivo the efficacy of several liposome and nonliposome formulations, including phospholipid liposomes and their cationic or pegylated variants, nonionic liposomes and their cationic variant, PINC polymer (Protective, Interactive, Noncondensing polymers), and a propylene glycol:alcohol:water mixture (delivery vehicle for minoxidil) in delivering beta-galactosidase and luciferase reporter genes into skin cells.

Identification of the cellular receptor for anthrax toxin.

The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol.

The little mutation suppresses DEN-induced hepatocarcinogenesis in mice and abrogates genetic and hormonal modulation of susceptibility.

In mice, the sex difference in susceptibility to hepatocarcinogenesis results from the tumor promoting activity of testosterone and from the inhibition of tumor promotion by ovarian hormones. We investigated the role of growth hormone in the sex-dependent regulation of susceptibility, because sex hormones are known to regulate the temporal pattern of growth hormone secretion and subsequent sex differences in liver gene expression. We found that in both males and females, wild-type mice developed significantly more tumors than growth hormone-deficient, C57BL/6J-lit/lit (B6-lit/lit) mutant mice following perinatal treatment with the carcinogen N,N-diethylnitrosamine (DEN).

Overexpression of an mRNA-binding protein in human colorectal cancer.

A Coding Region instability Determinant-Binding Protein (CRD-BP) binds in vitro to c-myc mRNA and appears to stabilize the mRNA. The CRD-BP gene is amplified in one-third of human breast cancer cases, and the CRD-BP appears to be an oncofetal protein. To analyse CRD-BP expression in human cancer tissue, paired extracts of cancer and normal colon specimens from 21 patients were analysed by immunoblotting and/or reverse transcriptase-polymerase chain reaction.

Defects in transcription coupled repair interfere with expression of p90(MDM2) in response to ultraviolet light.

Ultraviolet (UV) irradiation transiently stabilizes p53 through a mechanism that may require a decrease in the activity of the ubiquitin ligase, p90(MDM2). Conversely, the recovery of low levels of p53 following UV exposure may depend on an increase in p90(MDM2). The level of p90(MDM2) is increased by UV light following the p53-dependent induction of an internal mdm2 promoter, P2.

How sigma docks to RNA polymerase and what sigma does.

It is clear that multiple sites of interaction exist between sigmas and core subunits, likely reflecting the changing pattern of interactions that occur sequentially during the complex process of holoenzyme formation, open promoter formation, and initiation of transcription. Recent studies have revealed that a major site of interaction of Escherichia coli sigma factors is the amino acid 260-309 coiled-coil region of the beta' subunit of core RNA polymerase. This region of beta' interacts with region 2.

Interaction of the papillomavirus transcription/replication factor, E2, and the viral capsid protein, L2.

The minor capsid protein L2 of papillomaviruses (PVs) likely plays a role in the selective encapsidation of PV DNA in viral capsids and in the infectivity of PV virions. The L2 protein also can cause the relocalization of the PV early protein, E2TA, to nuclear subdomains known as promyelocytic leukemia oncogenic domains (PODs) in which it is localized. E2TA is a transcriptional transactivator that also plays a critical role in viral DNA replication.

EBNA-1: a protein pivotal to latent infection by Epstein-Barr virus.

Epstein-Barr nuclear antigen 1, or EBNA-1, is required for the replication of the EBV genome as an extra-chromosomal element and is a key transcriptional regulator of this virus's latent gene expression. In this review we will describe the salient features of EBNA-1 and oriP, the latent origin of EBV to which EBNA-1 binds site-specifically. EBNA-1's association with host cellular factors, its association with metaphase chromosomes, and its ability to link DNAs to which it binds will be discussed in relation to its roles in replication and transcriptional activation.

Human papillomavirus types 16 E6 and E7 contribute differently to carcinogenesis.

High-risk human papillomaviruses (HPVs) are etiologically implicated in human cervical cancer. Two viral genes, E6 and E7, are commonly found expressed in these cancer cells. We have previously shown that mice transgenic for the HPV-16 E6 gene or E7 gene, in which the E6 or E7 was expressed in the basal layer of epithelia, developed skin tumors.

Identification of three genes expressed primarily during development in Physarum polycephalum.

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA.

Establishment of the human papillomavirus type 16 (HPV-16) life cycle in an immortalized human foreskin keratinocyte cell line.

The study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult.

Identification of profilin and src homology 3 domains as binding partners for Drosophila enabled.

Drosophila Enabled (Ena) was first identified as a genetic suppressor of mutations in the Abelson tyrosine kinase and subsequently was shown to be a member of the Ena/vasodilator-stimulated phosphoprotein family of proteins. All members of this family have a conserved domain organization, bind the focal adhesion protein zyxin, and localize to focal adhesions and stress fibers. Members of this family are thought to be involved in the regulation of cytoskeleton dynamics.

Assays for analyzing exonucleases in vitro.

Ribonucleases play essential roles in cell growth, differentiation, and the response to stress. This article deals with exoribonucleases, enzymes that degrade RNAs beginning at either the 5' or 3' end and proceed down the length of the RNA. The preparation of a crude extract of a mammalian 3'-to-5' exonuclease is described.

Glucocorticoids stimulate CREB binding to a cyclic-AMP response element in the rat serine dehydratase gene.

Transcription of the rat serine dehydratase (SDH) gene, which is stimulated in hepatocytes by glucagon through the activity of the second messenger, cAMP, is augmented by pretreatment with glucocorticoids. A putative cAMP response element (CRE) located approximately 3.5 kbp upstream of the transcriptional start site was hypothesized to be responsible for this effect.

Targeting and retrofitting pre-existing libraries of transposon insertions with FRT and oriV elements for in-vivo generation of large quantities of any genomic fragment.

A procedure is described that converts the pre-existing transposon insertion libraries to a collection of 'pop-out' strains, each allowing generation of 20- to 100-kb genomic fragments directly from the genome. The procedure consists of two steps: (1) single transposon insertions are targeted and retrofitted with excision and amplification elements (FRT and oriV), by homologous recombination with an FRT-oriV-carrying plasmid; and (2) two retrofitted neighbouring transposons are brought together by P1 transduction. From each strain, a 20- to 100-kb genomic fragment, bound by a pair of retrofitted transposons, could be excised and amplified upon supplying in trans the excision (Flp) and replication (TrfA) functions.

The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins.

The half-life of c- myc mRNA is regulated when cells change their growth rates or differentiate. Two regions within c- myc mRNA determine its short half-life. One is in the 3'-untranslated region, the other is in the coding region.

Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways.

E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors in vivo. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis to determine how these two proteins modulate DNA damage responses in vivo.

The HIV-1 matrix domain of Gag is required for Vpu responsiveness during particle release.

HIV-1 viral protein U (Vpu) facilitates virus particle release. To determine whether Gag is sufficient for generation of a target for Vpu-mediated particle release, we expressed HIV-1 Gag protein in the absence of the other viral genes. The resulting particles were still Vpu responsive.

Functional antioxidant responsive elements.

Exposure of human and rodent cells to a wide variety of chemoprotective compounds confers resistance against a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes like glutathione S-transferases (GST). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system.