Transcript Document

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 9-40bp 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 mRNA 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
Transposome combines the advantages of in vivo and in vitro systems, it has
been developed utilizing the Tn5 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 TnAraOut 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 AraC 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 600bp 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 R6K origin of
replication (oriR6K).
• 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 IncP 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
(hdeA and yjbJ) 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 hdeA
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 mRNA
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 mRNAs; (2) translation initiation inhibition by
occlusion of the ribosome binding site; (3) premature termination of
mRNA transcription due to antisense binding to the genomic DNA
template; (4) stimulation of rapid mRNA 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 mRNA
inhibition using the Tcinducible shuttle vector
pYJ335 ( a ) The origins
of replication from pUC19
and pE194, 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 mRNA 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 mRNA is produced from
GAL1 promoter upon induction with
galactose. Plasmid integration through
homologous recombination can occur
either at the GAL1 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 mRNA expression
inhibition as well as saturating and signature-tagged mutagenesis
were designed to efficiently extract novel biological information
pertinent to an organism’s survival.