Richard A. Winegar

Transgenic Animal Models for Measuring Mutations In Vivo

Transgenic animal models for measuring mutations provide a powerful tool for rapidly assessing tissue-specific mutations following in vivo treatment. These models are based on the insertion into the rodent genome of the Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations. Following in vivo treatment of animals, genomic DNA is isolated from various tissues and the target gene is packaged into lambda-phage heads; the lambda-phage are used to infect E. coli in order to produce plaques. Mutations in the target gene are then detected using colorimetric or selective procedures. In this review methods are discussed for producing these transgenic models, the target genes used, gene rescue techniques, sequencing of isolated mutants, and parameters that affect dosing regimens and design of studies. We also present a summary of data published to date with these systems and present our conclusions and proposed directions for future research.

Alphaviruses: Population genetics and determinants of emergence

Alphaviruses are responsible for several medically important emerging diseases and are also significant veterinary pathogens. Due to the aerosol infectivity of some alphaviruses and their ability to cause severe, sometimes fatal neurologic diseases, they are also of biodefense importance. This review discusses the ecology, epidemiology and molecular virology of the alphaviruses, then focuses on three of the most important members of the genus: Venezuelan and eastern equine encephalitis and chikungunya viruses, with emphasis on their genetics and emergence mechanisms, and how current knowledge as well as gaps influence our ability to detect and determine the source of both natural outbreaks and potential use for bioterrorism. This article is one of a series in Antiviral Research on the genetic diversity of emerging viruses.

Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice.

Ionizing radiation induces gene mutations (point mutations, deletions and insertions) as well as chromosome damage in mammalian cells. Although these effects have been studied extensively in cells in culture, until recently it has not been possible to analyze the mutagenic potential of ionizing radiation in vivo, especially at the molecular level. The development of transgenic mutagenesis systems has now made it possible to study the effects of ionizing radiation at both the molecular and chromosomal levels in the same animal. In this report we present preliminary data on the response of Big Blue lacI transgenic mice to ionizing radiation as measured by lacI mutations and micronuclei. C57Bl/6 transgenic mice were irradiated with 137Cs gamma-rays at doses ranging from 0.1 to 14 Gy, and expression times ranging from 2 to 14 days. Dose-related increases in the mutant frequency were observed after irradiations with longer expression times. Mutant plaques were analyzed by restriction enzyme digestion to detect large structural changes in the target sequence. Of 34 gamma-ray-induced mutations analyzed, 4 were large-scale rearrangements. 3 of these rearrangements were deletions within the lacI gene characterized by the presence of short regions of homology at the breakpoint junctions. The fourth rearrangement was a deletion that extended from within the alpha lacZ gene into downstream sequences and that had 43 bp of homology at the junction. These data indicate that the Big Blue lacI transgenic mouse system in sensitive to the types of mutations induced by ionizing radiation. To determine whether the presence of the transgene affects micronucleus induction we compared the response of nontransgenic to hemizygous transgenic B6C3F1 mice and the response of nontransgenic to hemizygous and homozygous transgenic C57Bl/6 mice. The presence or absence of the lacI transgene had no effect on spontaneous micronucleus frequencies for either strain. However, radiation-induced micronucleus frequencies were significantly higher in hemizygous lacI B6C3F1 mice than in nontransgenic litter mates; the converse was true in C57Bl/6 mice. These data suggest that the lacI transgene does not cause chromosome instability as measured by spontaneous micronucleus levels. However, the response of these transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage.

Mechanisms involved in rejoining DNA double-strand breaks induced by ionizing radiation and restriction enzymes

DNA double-strand breaks are considered to be the most deleterious lesion induced by ionizing radiation. However, the mechanism of rejoining of these lesions has not been extensively studied at the molecular level. We have used a shuttle vector, pHAZE, to analyze the mechanism of rejoining of DNA double-strand breaks in human cells. The advantage of this vector system is that, unlike many previously described shuttle vectors, it has a large target gene for the detection of deletions and it is maintained as a freely replicating episome with chromatin conformation in the nucleus of human cells. In this study we compare data obtained on the spectrum of mutations induced in pHAZE by ionizing radiation (alpha-particles) and restriction enzymes (PvuII, ClaI, and PvuI). Unlike ionizing radiation, restriction enzymes induce double-strand breaks in DNA with known end structures at defined locations and therefore provide a model system for analyzing cellular responses to DNA double-strand breaks. Exposure of human cells containing the vector to alpha-particle irradiation produced both point mutations and large deletions in pHAZE. When the junction regions of the deletions were sequenced it was found that 65% were rejoined with up to 6 bp of homology at the junction region. Analysis of restriction-enzyme-induced mutations suggests that double-strand break ends are modified to facilitate rejoining and that the type of modification is characteristic for different end structures. Double-strand breaks with cohesive ends appear to have fewer modifications introduced at the break points before rejoining than breaks with blunt ends. When considered in relation to the data obtained with ionizing radiation this suggests that the presence of cohesive sequences either at, or in proximity to, the ends enhances rejoining of DNA double-strand breaks.

Modulation of Restriction Enzyme-Induced Damage by Chemicals That Interfere with Cellular Responses to DNA Damage: A Cytogenetic and Pulsed-Field Gel Analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.

Induction of chromosome damage by restriction enzymes during mitosis.

Once electroporated into the nucleus of eukaryotic cells, restriction enzymes will bind at specific DNA sequences and cleave DNA to make double-strand breaks. These induced breaks can lead to chromosome aberrations and consequently offer one approach to determining the mechanism(s) of aberration formation. Because the higher-order structure of DNA in eukaryotic cells might influence the ability of restriction enzymes to locate their recognition sequence, bind, and cleave DNA, we have investigated whether enzymes will cut DNA during metaphase when the chromosomes are most condensed. Chinese hamster ovary cells synchronized in mitosis and treated with either AluI or Sau3AI showed few chromosome aberrations when held in mitosis for 1, 2, or 3 h after enzyme treatment. However, some disruption of chromosome morphology was seen, especially after exposure to Sau3AI. When cells were allowed to complete one cell cycle after enzyme treatment in the preceding mitosis, there was extensive chromosome damage, with the most abundant type of lesion being the interstitial deletion. It appears that restriction enzymes will cleave the highly condensed DNA in mitotic cells but that decondensation, DNA replication, and recondensation are required before the aberrations are manifested.

Analysis of the mutagenic potential of ENU and MMS in germ cells of male C57BL/6 lacI transgenic mice

Mutant frequencies in male germ cells were determined in mice 3 days after exposure to saline, methylmethane sulfonate (MMS), or ethylnitrosourea (ENU). DNA was isolated from seminiferous tubules by a modified version of the drop dialysis method. A 5-fold increase in mutant frequency was observed in mice treated with ENU. No statistically significant increase was observed in mice treated with MMS.

Legal, Technical, and Interpretational Considerations in the Forensic Analysis of Viruses

The forensic evaluation of viruses presents new challenges to the forensic science community. Although many criminal cases have been adjudicated involving the deliberate transmission of viruses, especially HIV, this review provides a general approach to viral forensics, especially in light of significant biodefense challenges. Newly emerging techniques of nucleic acid sequencing are discussed in a forensic context. Human mitochondrial DNA analysis, wherein mixed profiles are routinely assessed in a forensic context, provides the groundwork for an interpretational approach to the issue of mixed DNA sequences. The importance of phylogenetic classification is discussed as both providing an integrated graphical depiction of the structure of viral nucleic acid variation as well as offering a tool that can be used to assess the relatedness of complex populations of nucleic acids.