Chapter 8 Mutagenesis As a Genomic Tool For

Chapter 8 Mutagenesis As a Genomic Tool For Studying Gene Function • Tian Baoxia • 2011. 04. 06

Introduction • Understanding the function of a particular gene is a multistep process. Initially, using bioinformatic tools to predict gene function. Second, measure gene and protein expression patterns. The third step in functional analysis involves system perturbation where the gene in question is inactivated. • In bacteria, mutagenesis is a relatively straightforward process as they are haploid organisms where any single mutation results in altered expression or impaired function. • In this chapter we will discuss the application of three major strategies for genome-wide mutagenesis: transposon insertion, gene disruption by allelic exchange, and expression inhibition using antisense RNA molecules.

Transposon Mutagenesis 1. Overview of transposition in bacteria A. classification of transposable elements There are two major groups in bacteria: insertion sequence (IS) and transposons (Tn) IS Contain two 9 -40 bp copies of terminally inverted nucleotide repeats The inverted repeats flank the transposase gene

Tn Have a central region carrying markers flanked by IS modules The IS arms are direct or inverted repeats Contains auxiliary genes unrelated to transposition

There are two major mechanisms for transposition: conservative and replicative transposition Replicative transposition Conservative transposition The conservative (or nonreplicative) mechanisms involves an excision of the transposon sequence from the donor molecule and subsequent insertion at the target site without duplication

B Insertion Specificity and Effects on Distal Gene Expression • Some transposons will preferentially insert into certain sites, others will avoid specific regions on the chromosome. transposon insertion patterns depend on both the frequency with which a particular site is used as a target and the detectability of the insertion. • Upon integration into the target site, transposons and insertion elements can affect the transcription of genes located in the vicinity of the insertion. Most often the transcriptional effects are due to terminator signals or polarity caused by long untranslated stretches of m. RNA when the insertion inactivates a gene (or genes) located in the beginning of an operon.

C Transposon Delivery Systems • A variety of delivery Vehicles have been developed , those include suicide phages and plasmids that are unable to replicate within the host strain, but possess mobilization ability and a broad host range of transfer. • Generally, use of phage delivery vehicles is restricted by the host specificity range and cannot be efficiently adapted for distantly related organisms that are not sensitive to bacteriophage infection. In contrast, plasmid vectors are more versatile with respect to transfer ability and can be used in a broader range of hosts. • In general, the choice of a delivery vehicle largely depends on the properties of the recipient strain and on the transposition target.

2. Transposons as Tools for Mutagenesis A. In vivo Mutagenesis • Advantage : The target organism does not have to be naturally competent • Disadvantage: The transposon must be introduced into the host on a suicide vector, the transposase must be expressed in the target host, and the transposase usually is expressed in subsequent generations, resulting in potential insertion instability

B. In vitro Mutagenesis The in vitro approach is based on the ability of purified transposases to catalyze strand-transfer reactions between linear DNA molecules in a cell-free environment Advantages: it have the ability to reach high saturation levels of mutagenesis, which allows one to conduct analyses of the target locus on either large or small scales. Disadvantage: it have the prerequisite for preliminary information on the target sequence.

Transposome combines the advantages of in vivo and in vitro systems, it has been developed utilizing the Tn 5 transposition system • Advantages: It overcomes the host barrier posed by in vivo transposition and the need for homologous recombination • Disadvantage: It is dependent on the availability of a transformation system

C. Advantages of Transposon Mutagenesis • Transposon mutagenesis results in the integration of foreign DNA into the target gene. The reversion frequency is lower than that of the single -base mutations. The excision of transposons from the target site usually occurs at low frequencies. • The transposition-related functions are provided by a cis-acting transposase located on the suicide donor molecule, which is lost following the transposition. • The selection marker of the transposon can be used to identify the size of the fragment of DNA that contains the transposon.

3. Transposon-based Approaches for Identification of Essential Genes There are two general strategies for the identification of essential genes: the negative approach and the positive approach. A. In vivo Global Transposon Mutagenesis Advantage: It can be applied to any sequenced organism that has a developed genetic system available Disadvantage: It requires a large number of transpson insertions

B. Genomic Analysis and Mapping by in vitro Transposition Advantage: The increased insertion frequency that makes the saturation of a particular genome region relatively easy Disadvantage: Its labor intensity and the large number of primers required for mapping

C. Mariner-based Tn. Ara. Out Transposon System Advantage: It is simple, cost-efficient, and can be adapted to a variety of bacteria that are permeable to arabinose and possess regulatory proteins of the Ara. C family of regulators.

4 Signature-tagged Mutagenesis for Studying Bacterial Pathogenicity Signature-tagged mutagenesis is a new transposon mutagenesis technique which utilizes a unique DNA sequence to tag each individual transposon molecule A. STM procedure First step: generate a library of tagged mutants Second step: screen the library

B. Common Considerations for Application of Signature-tagged Mutagenesis Generation of Mutant Libraries: • mutants was constructed by insertion-duplication mutagenesis • transposon molecule was replaced by randomly selected 400 - to 600 bp chromosomal fragments Hybridization Specificity: • select of 96 tagged transposons before mutagenesis • screening by hybridization was replaced by PCR detection with a set of reusable tags Pool Complexity: • limit the number of mutants that can be used in each pool Inoculum Dose and Administration: • high inoculum doses • an intraperitoneal administration is better than oral administration Duration of Infection: • long incubation times

Targeted mutagenesis through allelic exchange 1 Suicide Vector Systems for Allelic Exchange Suicide plasmid’s properties: • It is conditional for replication to allow selection for integration into the chromosome • It carry a selectable marker • It should be transferable to a wide variety of organisms Suicide Vector Systems： • One of the easiest strategies for identifying a suitable suicide vector involves utilization of the host range specificity for replication. • A robust suicide delivery system for targeted mutagenesis was developed utilizing plasmid vectors carrying the R 6 K origin of replication (ori. R 6 K). • The incorporation of counterselectable genes has been widely used for the construction of suicide vectors.

2 Strategies Commonly Utilized for Targeted Mutagenesis by Allelic Exchange A. Integration of Conditional Replicons by Single-cross-over Recombination: The Insertion–Duplication Method Drawbacks: First, the insertion polarity and internal promoter alter gene expression. Second, formation of two mutant copies of the target gene affect insertion stability and cause plasmid excision. Finally, selective pressure can affect cell fitness and result in phenotypes unrelated to the mutation.

B. Gene Replacement by Double-cross-over Recombination: The Deletion–Substitution Method Several other variations of the deletion–replacement method have been developed： • using plasmids of the Inc. P incompatibility groups • transform linear DNA substrates into the organism of interest. Disadvantage of PCR-based gene disruption: First, decrease efficiency of double-cross-over recombination which required to screen greater numbers of mutants. Second, polar effects are possible if there is a transcriptional terminator present in the cassette used for disruption.

C. Construction of Unmarked Deletions through Use of Counterselectable Markers Figure 8 Positive selection of allelic exchange mutants in a two-step selection strategy, using a counterselectable marker. ( a ) During the first step, an intermolecular recombination leads to the integration of the suicide vector carrying the mutated (deleted) allele. ( b ) The second step involves plasmid resolution through intermolecular recombination.

3. Application of Allele Exchange Approach in Functional Genomic Studies for Sequenced Microorganisms A. Characterization of Unknown Genes in E. coli Using In-frame Precise Deletions • PCR-based in-frame deletion system: Amplify target gene (hde. A and yjb. J) by PCR The resulting PCR products were placed in the E. coli chromosome by using a gene replacement vector Two genes proved to be nonessential Replace chromosomal hde. A with insertional alleles Essential and nonessential phenotypes were obtained • These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli.

B. Genome-wide Phenotypic Analysis of S. cerevisiae Mutants Using Molecular Bar-coding Strategy Deletion cassette Transform into a diploid yeast strain Cassette replace the coding sequence Km+, Molecular barcodes and homology sequence Mutant is analyzed by amplifying the barcodes Diploid strain sporulate and haploid segregant The molecular barcodes constitute 20 -bp sequences that are unique to each deletion and allow the identification of each deletion strain within a pool of many strains • Advantage: The comprehensive collection of null mutants can be screened for a specific phenotype. • Disadvantage: First, mutations in essential genes will not be represented in these haploid strains. Second, only annotated ORFs are deleted; Third, about 8% of the yeast deletion strains are aneuploid for a given chromosome region such that an extra copy of the gene may be retained by the cell. Finally, other mutations may exist as well.

Gene silencing using antisense m. RNA molecules 1. antisense RNA regulation in vivo • • Antisense RNAs are small, highly structured single-stranded molecules that act through sequence complementarity to inhibit target RNA (sense RNA) function. Mechanisms of regulation : (1) translation blockage by antisense hybridization to target m. RNAs; (2) translation initiation inhibition by occlusion of the ribosome binding site; (3) premature termination of m. RNA transcription due to antisense binding to the genomic DNA template; (4) stimulation of rapid m. RNA degradation by duplexspecific RNases; and (5) reduction of enzymatic activity by antisense binding to the target protein

2. Antisense Approach to Large-scale Functional Genomic Studies Genome-scale Antisense Silencing in S. aureus Using a Random Antisense RNA Library Figure 9 Antisense m. RNA inhibition using the Tc-inducible shuttle vector p. YJ 335 ( a ) The origins of replication from p. UC 19 and p. E 194, respectively, allowing plasmid replication in E. coli and S. aureus hosts. (b ) Upon addition of tetracycline or anhydrotetracycline, transcription from the xyl/tet promoter yields an antisense m. RNA of the target gene.

Gene Suppression in Candida albicans Using a Combination of Antisense Silencing and Promoter Interference Figure 10 Integration of antisense library plasmids into C. albicans genome. The gene X antisense m. RNA is produced from GAL 1 promoter upon induction with galactose. Plasmid integration through homologous recombination can occur either at the GAL 1 or the gene X genomic loci. In both cases, antisense RNA of gene X will be produced. Neutralization of a majority of sense RNA molecules transcribed from gene X occurs by hybridization with antisense RNA and leads to the translation of a reduced number of sense RNA molecules.

Summary • In this chapter, we focused on the application of gene inactivation methods applicable for genome-scale analysis that include transposon mutagenesis, gene disruption through allelic exchange, and gene silencing using antisense RNA. • Allelic replacement has proven to be a powerful method for determining gene function , however, the low throughput of this techniques makes them very difficult and impractical to perform on a genome-wide scale. Moreover, it makes the identification of genes essential for viability nearly impossible. • High throughput mutagenesis techniques such as m. RNA expression inhibition as well as saturating and signature-tagged mutagenesis were designed to efficiently extract novel biological information pertinent to an organism’s survival.

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