Transcript Recombination and Genetic Engineering

Recombination and
Genetic Engineering
Microbiology
Eucaryotic recombination

Recombination
– process in which one or
more nucleic acid
molecules are
rearranged or combined
to produce a new
nucleotide sequence

In eucaryotes, usually
occurs as the result of
crossing-over during
meiosis
Figure 13.1
Bacterial Recombination:
General Principles
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Several types of recombination
– General recombination
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can be reciprocal or nonreciprocal
– Site-specific recombination
– Replicative recombination
Reciprocal general
recombination
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Most common type of recombination
A reciprocal exchange between pair
of homologous chromosomes
Results from DNA strand breakage
and reunion, leading to crossing-over
Reciprocal
general
recombination
Figure 13.2
Figure 13.2
Nonreciprocal general
recombination
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Incorporation of
single strand of
DNA into
chromosome,
forming a stretch
of heteroduplex
DNA
Proposed to occur
during bacterial
transformation
Figure 13.3
Site-specific
recombination
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Insertion of nonhomologous DNA
into a chromosome
often occurs during viral genome
integration into host chromosome
– enzymes responsible are specific for
virus and its host
Site Specific
Recombination
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If the two sites undergoing
recombination are oriented in the
same direction, this may result in a
deletion
Inversions
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Recombination at inverted repeats causes
and inversion
Replicative recombination
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Accompanies replication of genetic
material
Used by genetic elements that move
about the genome
Horizontal gene transfer
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Transfer of genes from one mature,
independent organism (donor) to another
(recipient)
Exogenote
– DNA that is transferred to recipient
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Endogenote
– genome of recipient
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Merozyogote
– recipient cell that is temporarily diploid as
result of transfer process
Bacterial Plasmids
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Small, double-stranded, usually circular
DNA molecules
Are replicons
– have their own origin of replication
– can exist as single copies or as multiple copies
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Curing
– elimination of plasmid
– can be spontaneous or induced by treatments
that inhibit plasmid replication but not host cell
reproduction
Bacterial plasmids…
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Episomes
– plasmids that can exist either with or
without integrating into chromosome
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Conjugative plasmids
– have genes for pili
– can transfer copies of themselves to
other bacteria during conjugation
Fertility Factors
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conjugative
plasmids
e.g., F factor
of E. coli
many are also
episomes
Figure 13.5
F plasmid integration
mediated by
insertion sequences
(IS)
Figure 13.7
Resistance Factors
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R factors (plasmids)
Have genes for resistance to
antibiotics
Some are conjugative
usually do not integrate into
chromosome
Col plasmids
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Encode colicin
– kills E. coli
– a type of bacteriocin
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protein that destroys other bacteria,
usually closely related species
Some are conjugative
Some carry resistance genes
Other Types of Plasmids
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Virulence plasmids
– carry virulence genes
e.g., genes that confer resistance to host
defense mechanisms
 e.g., genes that encode toxins
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Metabolic plasmids
– carry genes for metabolic processes
e.g., genes encoding degradative enzymes for
pesticides
 e.g., genes for nitrogen fixation
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Transposable Elements
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Transposition
– the movement of pieces of DNA around
the genome
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Transposable elements (transposons)
– segments of DNA that carry genes for
transposition
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Widespread in bacteria, eucaryotes
and archaea
Types of transposable
elements
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Insertion sequences (IS elements)
– Contain only genes encoding enzymes required
for transposition
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Transposase
Composite transposons( Tn)
– Carry genes in addition to those needed for
transposition
– Conjugative transposons
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Carry transfer genes in addition to transposition
genes
IS sequences
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Insertion elements are mobile genetic elements that
occasionally insert into chromosomal sequences, often
disrupting genes .
Insertion elements are characterized by inverted terminal
repeats . These terminal repeats likely are recognition
sites for an enzyme responsible for the insertion.
Mobility of the element depends only on the element
itself; it is an autonomous element. Thus, it must carry the
coding ability for the transposase recognizing the inverted
terminal repeats.
The direct repeats externally flanking the inverted
repeats are not part of the insertion sequence. Instead,
they are chromosomal sequences that become duplicated
upon insertion, with one copy at each end; this is called
target-site duplication.
Characteristics of IS
elements
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The majority of IS elements are between 0.7 and l.8 kb in size
and the termini tend to be l0 to 40 base pairs in length with
perfect or nearly perfect repeats.
These sequences also tend to have RNA termination signals as
well as nonsense codons in all three reading frames and are
therefore polar.
Typically they encode one large open reading frame of 300 to
400 amino acids and by definition the protein encoded by this
reading frame is involved in the transposition event.
Two exceptions to the size range given above should be noted:
The first, }; is 5.7 kb and the other, IS101, is a scant 0.2 kb in
size. Although there are exceptions, insertion sequences tend to
be present in a small number of copies in the genome.
For example, IS1 is present in 6 to l0 copies in E. coli
chromosome while IS2 and 3 are typically present in about five
copies.
IS actions
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Insertion sequences mediate a variety of DNA
rearrangements. One of the first recognitions of
this fact was the involvement of insertion
sequences in the integration of F and R plasmids
into the host chromosome. This event gives rise
to Hfr strains.
The initial DNA rearrangement mediated by IS
elements is the "insertional duplication" that
they tend to generate at the site of insertion.
IS1 generates an 8 or 9 base pair duplication
while IS2 generates a 5 base pair duplication.
Transposons
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As defined above, a transposon is a mobile genetic
element containing additional genes unrelated to
transposition functions. In general, there are known to be
two general classes:
Class l or "compound Tns" encode drug resistance genes
flanked by copies of an IS in a direct or indirect repeat.
A direct repeat exists when the two sequences at either
end are oriented in the same direction while an indirect
(or inverted) repeat exists when they are in opposite
directions. In this class of transposons, the IS sequence
supplies the transposition function.
The second class of transposons are known as "complex"
or Class 2. With these, the element is flanked by short
(30-40 bp) indirect repeats with the genes for drug
resistance and transposition encoded in the middle (see
figure of Tn3 below).
Preferential sites for
transposition
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Class 1
GCTNAGC - Not AT rich
Sites found approximately every 100
bases in the E. coli genome
Class 2
AT rich regions are preferable sites
Homology at ends of region
The transposition event
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Usually transposon replicated,
remaining in original site, while
duplicate inserts at another site
Insertion generates direct repeats
of flanking host DNA
IR = inverted repeats
Figure 13.8
Tn3
transposition
Class 2 Transpoison
Complex Transposon
Generation
of direct
repeats
Effects of transposition
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Mutation in coding region
-deletion of genetic material
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Arrest of translation or
transcription
Activation of genes
Generation of new plasmids
– resistance plasmids
The U-tube experiment
after incubation,
bacteria plated on
minimal media
no prototrophs
Figure 13.13
demonstrated that
direct cell to cell
contact was
necessary
RTF = resistance
transfer factor
R1 plasmid
sources of
resistance genes
are transposons
a conjugative
plasmid
Bacterial Conjugation
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transfer of
DNA by direct
cell to cell
contact
discovered
1946 by
Lederberg and
Tatum
F+ x F– Mating
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F+ = donor
– contains F factor
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F– = recipient
– does not contain F factor
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F factor replicated by rolling-circle
mechanism and duplicate is transferred
recipients usually become F+
donor remains F+
F factor
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The F factor can exist in three different states:
F+ refers to a factor in an autonomous, extrachromosomal
state containing only the genetic information described
above.
The "Hfr" (which refers to "high frequency
recombination") state describes the situation when the
factor has integrated itself into the chromosome
presumably due to its various insertion sequences.
The F' or (F prime) state refers to the factor when it
exists as an extrachromosomal element, but with the
additional requirement that it contain some section of
chromosomal DNA covalently attached to it. A strain
containing no F factor is said to be "F-".
F+ x F– mating
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In its extrachromosomal
state the factor has a
molecular weight of
approximately 62 kb and
encodes at least 20 tra
genes. It also contains
three copies of IS3, one
copy of IS2, and one copy
of a À sequence as well as
genes for incompatibility
and replication.
Hfr Conjugation
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Hfr strain
– donor having F factor integrated into
its chromosome
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both plasmid genes and chromosomal
genes are transferred
Hfr x F– mating
Figure 13.14b
F Conjugation
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F plasmid
integrated F factor
– formed by
incorrect
excision from
chromosome
– contains  1
genes from
chromosome
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chromosomal gene
F cell can
transfer F
plasmid to
recipient
Figure 13.15a
F x
–
F
mating
Tra Y
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Characterization of the
Escherichia coli F factor traY gene
product and its binding sites
WC Nelson, BS Morton, EE Lahue
and SW Matson
Department of Biology, University of
North Carolina, Chapel Hill 27599.
Tra Genes
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Tra Y gene codes for the protein binds to
the Ori T
Initiates the transfer of plasmid across
the bridge between the two cells
Tra I Gene is a helicase responsible for
the conjugation
strand-specific transesterification
(relaxase)
Conjugative Proteins
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Key players are the proteins that
initiate the physical transfer of
ssDNA, the conjugative initiator
proteins
They nick the DNA and open it to
begin the transfer
Working in conjunction with the
helicases they facilitate the
transfer of ss RNA to the F- cell
DNA Transformation
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Uptake of naked DNA molecule from
the environment and incorporation
into recipient in a heritable form
Competent cell
– capable of taking up DNA
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May be important route of genetic
exchange in nature
Streptococcus pneumoniae
DNA binding
protein
competence-specific
protein
nuclease – nicks and degrades one
strand
Artificial transformation
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Transformation done in laboratory
with species that are not normally
competent (E. coli)
Variety of techniques used to make
cells temporarily competent
– calcium chloride treatment
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makes cells more permeable to DNA
Transduction
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Transfer of bacterial genes by
viruses
Virulent bacteriophages
– reproduce using lytic life cycle
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Temperate bacteriophages
– reproduce using lysogenic life cycle
Lysogenic Phage
Lambda
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In order for the lambda prophage to
exist in a host E. coli cell, it must
integrate into the host chromosome
which it does by means of a sitespecific recombination reaction.
Attachment site
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The E. coli chromosome contains one site at which lambda
integrates. The site, located between the gal and bio
operons, is called the attachment site and is designated
attB since it is the attachment site on the bacterial
chromosome.
The site is only 30 bp in size and contains a conserved
central 15 bp region where the recombination reaction will
take place.
he structure of the recombination site was determined
originally by genetic analyses and is usually represented as
BOB', where B and B' represent the bacterial DNA on
either side of the conserved central element
Recombination site
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The bacteriophage recombination site attP - is more complex. It contains the
identical central 15 bp region as attB.
The overall structure can be represented
as POP'. However, the flanking sequences
on either side of attP are very important
since they contain the binding sites for a
number of other proteins which are
required for the recombination reaction.
The P arm is 150 bp in length and the P'
arm is 90 bp in length.
Integration
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Integration of bacteriophage lambda requires
one phage-encoded protein - Int, which is the
integrase - and one bacterial protein - IHF,
which is Integration Host Factor.
Both of these proteins bind to sites on the P and
P' arms of attP to form a complex in which the
central conserved 15 bp elements of attP and
attB are properly aligned.
The integrase enzyme carries out all of the
steps of the recombination reaction, which
includes a short 7 bp branch migration.
Enzymes and
Recombination
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There are two major groups of enzymes that carry out sitespecific recombination reactions; one group - known as the
tyrosine recombinase family - consists of over 140 proteins.
These proteins are 300-400 amino acids in size, they contain two
conserved structural domains, and they carry out recombination
reactions using a common mechanism involving a the formation of
a covalent bond with an active site tyrosine residue.
The strand exchange reaction involves staggered cuts that are 6
to 8 bp apart within the recognition sequence.
All of the strand cleavage and re-joining reactions proceed
through a series of transesterification reactions like those
mediated by type I topoisomerases.
Excision of bacteriophages
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Excision of bacteriophage lambda requires two
phage-encoded proteins:
Int (again!) and Xis, which is an excisionase. It
also requires several bacterial proteins.
In addition to IHF, a protein called Fis is
required.
All of these proteins bind to sites on the P and
P' arms of attL and attR forming a complex in
which the central conserved 15 bp elements of
attL and attR are properly aligned to promote
excision of the prophage.
Generalized Transduction
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Any part of bacterial genome can be
transferred
Occurs during lytic cycle
During viral assembly, fragments of
host DNA mistakenly packaged into
phage head
– generalized transducing particle
Generalized transduction
Specialized Transduction
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also called restricted transduction
carried out only by temperate phages
that have established lysogeny
only specific portion of bacterial
genome is transferred
occurs when prophage is incorrectly
excised
Specialized
transduction
Figure 13.20
Figure 13.20
Mapping the Genome


locating genes on an organism’s
chromosomes
mapping bacterial genes
accomplished using all three modes
of gene transfer
Hfr mapping
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used to map relative location of bacterial
genes
based on observation that chromosome
transfer occurs at constant rate
interrupted mating experiment
– Hfr x F- mating interrupted at various intervals
– order and timing of gene transfer determined
Interrupted mating
Figure 13.22a
Figure 13.22b
E. coli genetic map
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gene locations
expressed in
minutes,
reflecting time
transferred
made using
numerous Hfr
strains
Figure 13.23
Transformation mapping
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used to establish gene linkage
expressed as frequency of
cotransformation
if two genes close together, greater
likelihood will be transferred on
single DNA fragment
Generalized transduction
mapping
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used to establish gene linkage
expressed as frequency of
cotransduction
if two genes close together, greater
likelihood will be carried on single
DNA fragment in transducing
particle
Specialized transduction
mapping
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provides distance of genes from
viral genome integration sites
viral genome integration sites must
first be mapped by conjugation
mapping techniques
Recombination and Genome
Mapping in Viruses
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viral genomes can also undergo
recombination events
viral genomes can be mapped by
determining recombination frequencies
physical maps of viral genomes can also be
constructed using other techniques
Recombination mapping

recombination
frequency
determined
when cells
infected
simultaneously
with two
different
viruses
Figure 13.24
Physical maps

heteroduplex maps
– genomes of two different viruses
denatured, mixed and allowed to anneal
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regions that are not identical, do not
reanneal
– allows for localization of mutant alleles
Physical maps…
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restriction endonuclease mapping
– compare DNA fragments from two
different viral strains in terms of
electrophoretic mobility

sequence mapping
– determine nucleotide sequence of viral
genome
– identify coding regions, mutations, etc.