© Borgis - New Medicine 4/2012, s. 116-121
*Paweł Kowalczyk1, Karol Chalimoniuk2, Agnieszka Danielak2, Dorota Dziedziela2, Paulina Jankowska2, Marzena Kowalska2, Joanna Laskowska2, Marzena Rachocka2, Jarosław Szczepaniak2, Tomasz Walter2, Paulina Strzyga2, Justyna Szymańska2, Mariusz Słomka2, Katarzyna Zawadka2, Martyna Staszewska2
M13mp18 phage model as a tools of research mutagenic and cytotoxic biological and environmental compounds
1Autonomous Department of Microbial Biology, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Poland
Head of Department: assoc. prof. Małgorzata Łobocka, MD, PhD
2Scientific Circle of Molecular Biology, Autonomous Department of Microbial Biology, Faculty of Agriculture and Biology, Warsaw University of Life Sciences
Introduction. Vectors of the mp series are all derived from a recombinant M13 bacteriophage (M13mp1) that carries a short segment of E. coli DNA in its major intergenic region. This segment contains the regulatory sequences and the coding information for the first 146 amino acids of the β-galactosidase gene (lacZ). M13 is an ideal vector for obtaining single-stranded templates required for the dideoxynucleotide chain termination method of sequencing DNA and site-directed mutagenesis using synthetic oligonucleotides.
Aim. M13 phage is a usefull tools for detection of different chemical environmental compounds such as: vinyl chloride (CV), chloroacetaldehyde (CAA) and other alkylating and oxidative agents e.g. trans-4-hydroxy-2-nonenal (HNE).
Material and methods. This vector allow estimate how strong is the chemical compound; by measure the level of survival and mutation frequency in the after transfection into bacterial strains after modification of phage DNA by different compounds concentration.
Results. Modification of single stranded M13 phage DNA with HNE may inhibit in vitro DNA synthesis by T7 DNA polymerase. The most reactive base within the M13 lacZ gene was 63% of guanine residues within the examined sequence), followed by cytosine (55%), adenine (39%), and finally thymine (25%).
Conclusions. The long chain HNE adducts to DNA bases arrest DNA synthesis to all four DNA bases and cause recombination, base substitutions and frameshift mutations in ssDNA and may constitute a strong hindrance for DNA synthesis. This DNA modification may lead to mutation and finally induce tumor progression.
Lac Z Operon
The lac operon is composed of two distinct regions of DNA. These are typically known as the LacI region and the LacZ regions. The LacI region is responsible for controlling the production of ß-galactosidase, the key enzyme that can break down lactose into glucose and galactose. As the LacI region is a gene, it codes for a specialized protein known as a repressor. The LacZ region consists of three seperate gene sequences: lacA, lacY and lacZ (Fig. 1). The lacA gene codes for transacetylase, which seemingly has no role in this system. The lacY gene codes for a lactose permease, which facilitates the movement of lactose into the cell membrane from the outside environment. The lacZ gene codes the ß-galactosidase. This enzyme hydrolyzes the bond between the two sugars, glucose and galactose. If lactose is not available in the medium, the genes for metabolizing the sugar are not expressed. However, if lactose is present in the medium, the genes for metabolizing this sugar are expressed and the bacterium is able to use lactose as an energy source. Preceding each gene region is a control sequence of DNA known as a promoter. In addition to this promoter, the LacZ region also contains an additional region known as an operator (fig. 1).
Fig. 1. Schematic presentations of the lac operon.
M13 as a cloning vector and a model for studies of mutations
The lacZ operon has been incorporated into several cloning vectors, specific to E. coli including filamentous bacteriophage e.g. M13. Is a filamentous, male-specific coliphage composed of a single-stranded closed circular molecule of DNA approximately 6400 nucleotides in length and a protein coat. Because of its filamentous structure, additional DNA can be inserted into its genome and packaged into an extended phage capsid. During infection, the phage attach only to the sex pili (conjugation tube) encoded by the F episome and only male bacteria are used to propagate the virus. Bacterial strains carrying F’ episomes and a number of genetic markers useful in work with M13 vectors have been constructed by Messing (1). The most important of these markers are: lacZ?M15A deletion mutant lacking the sequences of the lacZ gene coding for the amino-terminal portion of β-galactosidase (2). The peptide expressed by ?M15 can take part in α-complementation JM105 E. coli strain, a host for bacteriophage M13 vectors carries this deleted version of the lacZ gene on an F’ episome. In phage particles the chromosome of phage exists as a single-stranded circular DNA and is converted by cellular enzymes into a double-stranded circular form, called replicative form (RF). The replicative form replicates and gives rise to single stranded (the + strand) molecules which are packaged into phage and extruded from the cell. M13 does not lyse its host; rather, the phage particles containing single-stranded DNA are continuously secreted into the media by the cells.
Vectors of the mp series are all derived from a recombinant M13 bacteriophage (M13mp1) that carries a short segment of E. coli DNA in its major intergenic region. This segment contains the regulatory sequences and the coding information for the first 146 amino acids of the β-galactosidase gene (lacZ). Messing and others (1) further modified the phage M13mp1 by creating an EcoRI restriction site within the gene encoding β-galactosidase and added different other a useful series of densely packed restriction sites into the lacZ, and created the M13 vectors known as M13mp18. This phage (M13mp18) is easily screened for the introduction of a foreign DNA fragment by assaying for β-galactosidase activity in appropriate E. coli hosts. The properties described above make M13 an ideal vector for obtaining single-stranded templates required for the dideoxynucleotide chain termination method of sequencing DNA and site-directed mutagenesis using synthetic oligonucleotides. The M13mp18 genes are shown (fig. 2) on the map (M13 genes are transcribed clockwise).
Fig. 2. Development of M13 into a cloning vector.
M13 was developed into a useful cloning vector by inserting the following elements into the genome: a gene for the lac repressor (lacI) protein to allow regulation of the lac promoter: the operator-proximal region of the lacZ gene (to allow for α-complementation in a host with operator-proximal deletion of the lacZ gene): a lac promoter upstream of the lacZ gene; a polylinker (multiple cloning site) region inserted several codons into the lacZ gene.
This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic (α-complementation with a defective form of beta-galactosidase encoded by host (mutation lacZ?M15). The defective β-galactosidase protein produced by the F episome can complement the defective β-galactosidase encoded on the M13 vector (even with the presence of the polylinker). Ligation of DNA into the polylinker insertionally inactivates the vector-based defective lacZ gene product, disrupting the “α-complementation” leading to the loss of β-galactosidase activity. Thus the plaques retain their normal white color on X-gal/IPTG indicator plates. Screening for “white” plaques amongst the “blue” ones allows selection for DNA molecules inserted into the vector. However, one should be aware that almost any change in the polylinker (such as a disruption of one of the restriction sites) will also give “white” plaques, as will cells which have lost the F’ episome (when selection is relaxed). IPTG in the lacZ gene induces production of the functional galactosidase which cleaves X-Gal and results in a blue colored metobolite. The usual substrate for the lacZ gene protein product is lactose, which is metabolized into galactose and glucose. X-Gal is a colorless, modified galactose sugar, which, when hydrolysed by β-galactosidase yields blue phage plaques. M13 phage system is also an ideal forward mutation system, which can detect base substitutions and frameshits within M13 lacZ gene fragment, that effect β-galactosidase activity and in consequence α-complementation. Mutant phages defective in β-galactosidase can be thus easily identified as white or light blue plaques among wild type dark blue ones. The system enables also to monitor recombination events between lacZ gene positioned in the phage DNA and the host F’ factor, which appear as double deletion of large lacZ gene fragments – a 93 nucleotides correspoding to ?M15, and a 54 nucleotides, which corresponds to polylinker, cloned into M13 vectors.
The aim of study was to provide a broad database on formation, repair and genotoxic properties of exocyclic DNA adducts. These types of DNA lesions may be induced both by lipid peroxidation (LPO). LPO produces a family of propano-type and etheno-type DNA adducts usually substituted with alkyl chain of different length. The identity of DNA lesions, as well as proteins engaged in their processing are not well elucidated. I intended to investigate the consequences and sequence specificity of interaction with DNA of two types of compounds introducing into DNA exocyclic DNA adducts: (i) one of the major LPO product, trans-4-hydroxy-2-nonenal (HNE). Although HNE is abundant in tissues and plasma of rodents and humans, the molecular characterization of its adducts to DNA bases is still limited. This prompted me to address questions concerning: (I) the identity of HNE adducts to DNA bases by the effect on DNA synthesis in vitro and in transformation into E. coli cells; (II) their mutagenic properties in M13 phage system in lacZ gene.
Acrylamide, bisacrylamide, calcium chloride, chloroform, PEG, phenol, set of four individual dNTPs and Tris were purchased from Sigma. DNA sequencing kit (T7 Sequencing Kit), [α-S35]ATP (1000 Ci/mmol) and [γ-32P]ATP (3000 Ci/mmol) were from Amersham-Pharmacia Biotech or ICN. IPTG was from Promega. X-gal were obtained from MP Biomedicals.
The compound trans-4-hydroxy-2-nonenal (HNE) was synthesized in the form of a dimethylacetal derivative according to Chandra and Srivastava (6) with minor modifications with prof. Kuśmierek group of IBB PAS. The enzymes: T7 polymerase (Sequenase version 2.0) was obtained from Amersham, Tag polymerase was obtained from Promega.
Purification of M13mp18 phage DNA
Bacteriophage M13mp18 was grown overnight at 37°C in JM 105 strain of E. coli in 2YT medium (3). Phage particles were precipitated from the medium with polyethylene glycol, and DNA was isolated by the phenol/ /chloroform method as described by Messing (1).
Modification of M13mp18 phage DNA by e.g. HNE
Single-stranded M13 phage DNA was incubated with different concentrations of HNE at pH 5.5 for 2 h at 37°C. For each HNE concentration, as well as for the untreated control, 20 μg of phage DNA in a total volume of 100 μl was used. Subsequently, the DNA was ethanol precipitated and resuspended in 100 μl of sterile water.
Preparation of competent cells and transformation
Bacteria were grown at 37°C in LB medium and made competent by the CaCl2 method (3). Transfection was performed according to Sambrook (3) with 100 ng phage DNA used to transfect 100 μl of competent cells. Transfection mixtures were plated on LB solid medium with 3 ml of LB soft agar suplemented with 0.4 mM IPTG and 0.5 mg/ml X-gal. Plates were incubated overnight at 37°C and plaques of phage infective centers were scored.
Collection and sequencing of lacZ mutants
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