In vivo analysis of the 3′ untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone

Large sections of the 3′ untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3′ terminal conserved region or the poly(U–UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo. However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem–loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3′ UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U–UC) region and the conserved region, but not the variable region, of the 3′ UTR seem to be critical for in vivo infectivity of HCV.

The active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions

HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.

Mu in vitro Transposition Technology in Functional Genetics and Genomics: Applications on Mouse and Bacteriophages

Transposons are mobile elements of genetic material that are able to move in the genomes of their host organisms using a special form of recombination called transposition. Bacteriophage Mu was the first transposon for which a cell-free in vitro transposition reaction was developed. Subsequently, the reaction has been refined and the minimal Mu in vitro reaction is useful in the generation of comprehensive libraries of mutant DNA molecules that can be used in a variety of applications. To date, the functional genetics applications of Mu in vitro technology have been subjected to either plasmids or genomic regions and entire genomes of viruses cloned on specific vectors. This study expands the use of Mu in vitro transposition in functional genetics and genomics by describing novel methods applicable to the targeted transgenesis of mouse and the whole-genome analysis of bacteriophages. The methods described here are rapid, efficient, and easily applicable to a wide variety of organisms, demonstrating the potential of the Mu transposition technology in the functional analysis of genes and genomes. First, an easy-to-use, rapid strategy to generate construct for the targeted mutagenesis of mouse genes was developed. To test the strategy, a gene encoding a neuronal K+/Cl- cotransporter was mutagenised. After a highly efficient transpositional mutagenesis, the gene fragments mutagenised were cloned into a vector backbone and transferred into bacterial cells. These constructs were screened with PCR using an effective 3D matrix system. In addition to traditional knock-out constructs, the method developed yields hypomorphic alleles that lead into reduced expression of the target gene in transgenic mice and have since been used in a follow-up study. Moreover, a scheme is devised to rapidly produce conditional alleles from the constructs produced. Next, an efficient strategy for the whole-genome analysis of bacteriophages was developed based on the transpositional mutagenesis of uncloned, infective virus genomes and their subsequent transfer into susceptible host cells. Mutant viruses able to produce viable progeny were collected and their transposon integration sites determined to map genomic regions nonessential to the viral life cycle. This method, applied here to three very different bacteriophages, PRD1, ΦYeO3 12, and PM2, does not require the target genome to be cloned and is directly applicable to all DNA and RNA viruses that have infective genomes. The method developed yielded valuable novel information on the three bacteriophages studied and whole-genome data can be complemented with concomitant studies on individual genes. Moreover, end-modified transposons constructed for this study can be used to manipulate genomes devoid of suitable restriction sites.

Mu in vitro transposition applications in protein engineering

Transposons, mobile genetic elements that are ubiquitous in all living organisms have been used as tools in molecular biology for decades. They have the ability to move into discrete DNA locations with no apparent homology to the target site. The utility of transposons as molecular tools is based on their ability to integrate into various DNA sequences efficiently, producing extensive mutant clone libraries that can be used in various molecular biology applications. Bacteriophage Mu is one of the most useful transposons due to its well-characterized and simple in vitro transposition reaction. This study establishes the properties of the Mu in vitro transposition system as a versatile multipurpose tool in molecular biology. In addition, this study describes Mu-based applications for engineering proteins by random insertional transposon mutagenesis in order to study structure-function relationships in proteins. We initially characterized the properties of the minimal Mu in vitro transposition system. We showed that the Mu transposition system works efficiently and accurately and produces insertions into a wide spectrum of target sites in different DNA molecules. Then, we developed a pentapeptide insertion mutagenesis strategy for inserting random five amino acid cassettes into proteins. These protein variants can be used especially for screening important sites for protein-protein interactions. Also, the system may produce temperature-sensitive variants of the protein of interest. Furthermore, we developed an efficient screening system for high-resolution mapping of protein-protein interfaces with the pentapeptide insertion mutagenesis. This was accomplished by combining the mutagenesis with subsequent yeast two-hybrid screening and PCR-based genetic footprinting. This combination allows the analysis of the whole mutant library en masse, without the need for producing or isolating separate mutant clones, and the protein-protein interfaces can be determined at amino acid accuracy. The system was validated by analysing the interacting region of JFC1 with Rab8A, and we show that the interaction is mediated via the JFC1 Slp homology domain. In addition, we developed a procedure for the production of nested sets of N- and C-terminal deletion variants of proteins with the Mu system. These variants are useful in many functional studies of proteins, especially in mapping regions involved in protein-protein interactions. This methodology was validated by analysing the region in yeast Mso1 involved in an interaction with Sec1. The results of this study show that the Mu in vitro transposition system is versatile for various applicational purposes and can efficiently be adapted to random protein engineering applications for functional studies of proteins.

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG(20)k-S7C, PEG(20)k-K173C, and PEG(20)k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life. (C) 2004 Elsevier Inc. All rights reserved.

Microsatellite analysis for determination of the mutagenicity of extremely low-frequency electromagnetic fields and ionising radiation in vitro.

Extremely low-frequency electromagnetic fields (ELF-EMF) have been reported to induce lesions in DNA and to enhance the mutagenicity of ionising radiation. However, the significance of these findings is uncertain because the determination of the carcinogenic potential of EMFs has largely been based on investigations of large chromosomal aberrations. Using a more sensitive method of detecting DNA damage involving microsatellite sequences, we observed that exposure of UVW human glioma cells to ELF-EMF alone at a field strength of 1 mT (50 Hz) for 12 h gave rise to 0.011 mutations/locus/cell. This was equivalent to a 3.75-fold increase in mutation induction compared with unexposed controls. Furthermore, ELF-EMF increased the mutagenic capacity of 0.3 and 3 Gy gamma-irradiation by factors of 2.6 and 2.75, respectively. These results suggest not only that ELF-EMF is mutagenic as a single agent but also that it can potentiate the mutagenicity of ionising radiation. Treatment with 0.3 Gy induced more than 10 times more mutations per unit dose than irradiation with 3 Gy, indicating hypermutability at low dose.

In vitro isoflavone supplementation reduces hydrogen peroxide-induced DNA damage in sperm

Isoflavones are plant compounds, proposed to have health benefits in a variety of human diseases, including coronary heart disease and endocrine-responsive cancers. Their physiological effects include possible antioxidant activity, therefore suggesting a role for isoflavones in the prevention of male infertility. The aim of this study was to test the antioxidant effects of the isoflavones genistein and equol on sperm DNA integrity, assessed in vitro after hydrogen peroxide-mediated damage, using the cornet assay. Pre-treatment with genistein or equol at doses of 0.01-100 mumol/l significantly protected sperm DNA against oxidative damage. Both ascorbic acid (10-600 mumol/l) and alpha-tocopherol (1-100 mumol/l) also protected. Compared with ascorbic acid and alpha-tocopherol, added at physiological concentrations, genistein was the most potent antioxidant, followed by equol, ascorbic acid, and alpha-tocopherol. Genistein and equol added in combination were more protective than when added singly. Based on these preliminary data, which are similar to those observed previously in lymphocytes, these compounds may have a role to play in antioxidant protection against male infertility.

Human chromosome aberrations induced in vitro by Paracoccidioides brasiliensis glycoproteic component (GP 43)

The in vitro cytogenetic effects of the 43-kDa molecular mass exocellular glycoproteic component (GP 43) from Paracoccidioides brasiliensis were studied in cultures from human lymphocytes. The sample included 10 healthy, white, non-smoking, non-related males (mean age of 31.3 ± 8.2 years). Besides the control, three concentrations of GP 43 (0.125, 1.25 and 5 μg/ml) were used. In each group, around 1000 cells were examined in search of chromosome aberrations, and 30,000 metaphases were analysed for the determination of the Mitotic Index. The authors conclude that GP 43 most probably causes inhibition of the cell cycle and aneugenic and clastogenic effects.

Antimicrobial endodontic compounds do not modulate alkylation-induced genotoxicity and oxidative stress in vitro

Objective: To investigate if formocresol, paramonochlorophenol, or calcium hydroxide modulate the genotoxic effects induced by the oxidatively damaging agent hydrogen peroxide (H 2O 2) or the alkylating agent methyl methanesulfonate (MMS) in vitro by using single cell gel (comet) assay. Study design: Chinese hamster ovary (CHO) cells in culture were exposed directly to formocresol, paramonochlorophenol, or calcium hydroxide (adjusted to 100 μg/mL) for 1 hour at 37°C. Subsequently the cultures were incubated with increasing concentrations (0-10 μmol/L) of MMS in phosphate-buffered solution (PBS) for 15 minutes at 37°C or of H 2O 2 at increasing concentrations (0-100 μmol/L) in distilled water for 5 minutes on ice. The negative control cells were treated with PBS for 1 hour at 37°C. The parameter from the comet assay (tail moment) was assessed by the Kruskal-Wallis nonparametric test followed by a post hoc analysis (Dunn test). Results: Clear concentration-related effects were observed for the genotoxin-exposed CHO cells. Increase of MMS-induced DNA damage was not significantly altered by the presence of the compounds tested. Similarly, no significant changes were observed when hydrogen peroxide was used with the endodontic compounds evaluated. Conclusion: Formocresol, paramonochlorophenol, and calcium hydroxide are not able to modulate alkylation-induced genotoxicity or oxidative DNA damage as depicted by the single cell gel (comet) assay. © 2006 Mosby, Inc. All rights reserved.

Ação moduladora do citral e eugenol em eventos genéticos em magrófagos murinos in vitro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo do potencial genotóxico da Gutiferona A em diferentes células de camundongos in vitro

Pós-graduação em Biologia Geral e Aplicada - IBB

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

The cosmetic dye quinoline yellow causes DNA damage in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)