Helmut Zarbl

Transcriptional and Posttranscriptional Silencing of Rodent α1(I) Collagen by a Homologous Transcriptionally Self-Silenced Transgene

ABSTRACT Transient transfection of rodent fibroblasts with plasmids carrying a full-size pro-α1(I) collagen gene (pWTC1) results in rapid reduction of the endogenous transcripts by >90%, while the transgene mRNA is undetectable. Using deletion constructs, two adjacent 5′ noncoding regions of the gene are identified as causing transcriptional silencing of the endogene in normal and v-fos-transformed cells but not in nontumorigenic revertants, which show partial relief from v-fos transformation-induced α1(I) gene suppression. The 3′ end of the transgene carries an additional element(s), causing posttranscriptional silencing of the endogene in all cells including the revertant. Data indicate that the transgenes are transcriptionally self-silenced. Genome-integrated transgenes that are transcriptionally active also allow expression of the endogene, suggesting gene activation by chromosomal factors missing in pWTC1. Silencing is not regulated by antisense RNA. Silencing of the endogenous pro-α1(I) collagen gene is not linked to the level of transgene expression.

The Reovirus Multiplication Cycle

Human reoviruses have been recovered from a variety of mammals (Stanley, 1961a; Rosen, 1968). Serological studies, which reveal the presence of reovirus specific antibodies, have indicated a ubiquitous distribution of reovirus among mammals (Stanley, 1961a, b; Rosen, 1962). The American Type Culture Collection has suggested at least 14 mammalian cell lines as suitable for the growth of reoviruses, including cell lines derived from 12 different species and 7 different tissue types (see Catalogue of Strains II, 3rd ed., 1981). As a result of this extensive host range, several aspects of the reovirus multiplication cycle have been studied at one time or another in a variety of mammalian cells. However, most of the studies concerning the molecular biology of the reovirus multiplication cycle have been carried out on mouse L fibroblasts infected with the Dearing strain of reovirus type 3. The reason for this is that mouse L cells grow readily in suspension culture and that the Dearing strain multiplies particularly well in these cells. Therefore, the discussion of the events in the reovirus multiplication cycle will of necessity be based primarily on studies of reovirus multiplication in mouse L cells. Wherever appropriate, the discussion will include the multiplication of all three serotypes of human reovirus in a variety of cell lines.

Regulation of translation in L-cells infected with reovirus☆

Abstract It was reported recently that reovirus progeny subviral particles contain an active transcriptase (RNA polymerase) enzyme and inactive or masked guanylyl transferase and methylase enzymes. Another recent report showed that the translational machinery of L-cells, which is normally “cap” dependent, becomes modified and undergoes a transition to cap independence as a function of time post-infection. In the present work we have examined the 5′-terminal structure of messenger RNA associated with polyribosomes of L-cells infected with reovirus at early, intermediate and late times post-infection. The results show that reovirus mRNA molecules isolated from polyribosomes at early times contain predominantly, the 5′-terminal structure m7G(5′)ppp(5′)G(m)pCp … or type-1 cap; mRNA isolated at intermediate times contains two types of mRNA molecules, one terminating in a type-1 cap and the other having an uncapped 5′ terminus whose structure is pG …; at late times, essentially all viral mRNA is mono-phosphorylated at the 5′ terminus and is therefore uncapped. This pG …-terminated mRNA is not a substrate for the host capping enzymes, it cannot be translated in vitro in extracts from uninfected cells, but can be translated with high efficiency in extracts prepared from infected cells. L-cell mRNA molecules present in infected cells at late times do not seem to be degraded (at least not extensively) and can be translated in vitro in extracts from uninfected L-cells. These and other results are consistent with the idea that reovirus induces a gradual modification in the cap dependence of the host translational apparatus. As a result of this modification, uncapped reovirus mRNA is translated preferentially over host (capped) mRNA. These data have been drawn together in the form of a revised model for the reovirus replicative cycle.

Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage.

ABSTRACT Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB1-induced DNA damage in the yeast model. Rev3 appears to mediate AFB1-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB1-induced mutagenicity.

The Cycad Genotoxin MAM Modulates Brain Cellular Pathways Involved in Neurodegenerative Disease and Cancer in a DNA Damage-Linked Manner

Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O 6-methyldeoxyguanosine lesions, O 6-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O 6-mG DNA methyltransferase (MGMT) showed elevated O 6-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimers disease.

Reovirus core particles synthesize capped oligonucleotides as a result of abortive transcription

Abstract Reovirus cores were found to synthesize oligonucleotides concomitantly with mRNA under conditions of optimal transcription in vitro . These oligonucleotides resembled the major virion-associated 5′-G-terminated oligonucleotides, except that they contained the 5′ terminal cap structures m7GpppGp … and m7GpppGmpCp. … The capped oligonucleotides were not derived by modification of endogenous virion-associated oligonucleotides; rather, they were synthesized de novo . Capped oligonucleotides were synthesized in a standard transcriptase reaction with linear kinetics by the same particles which synthesize capped mRNA. The effect of altering the rate of RNA chain elongation indicated that capped oligonucleotides are the results of premature release of nascent transcripts.

DNA Microarrays: An Overview of Technologies and Applications to Toxicology

DNA microarrays or chips can be used to simultaneously monitor the expression levels and/or genotypes of thousands of genes. The application of these techniques heralds a new era in toxicology research, where genotypes and toxicant‐induced expression signatures may be used to monitor cellular responses to different doses, to classify toxins on the basis of their mechanisms of action, to monitor exposures, and to predict individual variability in toxicant sensitivity. This unit reviews the current state of microarray technologies and discusses potential applications in toxicology, with emphasis on the strengths and limitations of the technologies.

Patterns of gene expression in mammary cells following carcinogen exposure of strains of rat sensitive or resistant to carcinogenesis

Patterns of gene expression in mammary cells following carcinogen exposure of strains of rat sensitive or resistant to carcinogenesis