Recombination and Genetic Engineering

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Recombination and Genetic Engineering

• Microbiology

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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
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Bacterial Recombination General Principles

• Several types of recombination
• General recombination
• can be reciprocal or nonreciprocal
• Site-specific recombination
• Replicative recombination

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Reciprocal general recombination

• Most common type of recombination
• A reciprocal exchange between pair of homologous
  chromosomes
• Results from DNA strand breakage and reunion,
  leading to crossing-over

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Reciprocal general recom-bination
Figure 13.2
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Figure 13.2
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Nonreciprocal general recombination

• Incorporation of single strand of DNA into
  chromosome, forming a stretch of heteroduplex DNA
• Proposed to occur during bacterial transformation

Figure 13.3
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Site-specific recombination

• 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

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Site Specific Recombination

• If the two sites undergoing recombination are
  oriented in the same direction, this may result
  in a deletion

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Inversions

• Recombination at inverted repeats causes and
  inversion

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Replicative recombination

• Accompanies replication of genetic material
• Used by genetic elements that move about the
  genome

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Horizontal gene transfer

• Transfer of genes from one mature, independent
  organism (donor) to another (recipient)
• Exogenote
• DNA that is transferred to recipient
• Endogenote
• genome of recipient
• Merozyogote
• recipient cell that is temporarily diploid as
  result of transfer process

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Bacterial Plasmids

• Small, double-stranded, usually circular DNA
  molecules
• Are replicons
• have their own origin of replication
• can exist as single copies or as multiple copies
• Curing
• elimination of plasmid
• can be spontaneous or induced by treatments that
  inhibit plasmid replication but not host cell
  reproduction

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Bacterial plasmids

• Episomes
• plasmids that can exist either with or without
  integrating into chromosome
• Conjugative plasmids
• have genes for pili
• can transfer copies of themselves to other
  bacteria during conjugation

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Fertility Factors

• conjugative plasmids
• e.g., F factor of E. coli
• many are also episomes

Figure 13.5
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F plasmid integration
mediated by insertion sequences (IS)
Figure 13.7
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Resistance Factors

• R factors (plasmids)
• Have genes for resistance to antibiotics
• Some are conjugative
• usually do not integrate into chromosome

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Col plasmids

• Encode colicin
• kills E. coli
• a type of bacteriocin
• protein that destroys other bacteria, usually
  closely related species
• Some are conjugative
• Some carry resistance genes

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Other Types of Plasmids

• Virulence plasmids
• carry virulence genes
• e.g., genes that confer resistance to host
  defense mechanisms
• e.g., genes that encode toxins
• 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

• Transposition
• the movement of pieces of DNA around the genome
• Transposable elements (transposons)
• segments of DNA that carry genes for
  transposition
• Widespread in bacteria, eucaryotes and archaea

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Types of transposable elements

• Insertion sequences (IS elements)
• Contain only genes encoding enzymes required for
  transposition
• Transposase
• Composite transposons( Tn)
• Carry genes in addition to those needed for
  transposition
• Conjugative transposons
• Carry transfer genes in addition to transposition
  genes

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IS sequences

• 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.

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Characteristics of IS elements

• 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.

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IS actions

• 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.

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Transposons

• 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).

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Preferential sites for transposition

• 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

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The transposition event

• Usually transposon replicated, remaining in
  original site, while duplicate inserts at another
  site
• Insertion generates direct repeats of flanking
  host DNA

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IR inverted repeats
Figure 13.8
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Tn3 trans-position
Class 2 Transpoison Complex Transposon
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Generation of direct repeats
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Effects of transposition

• Mutation in coding region
• -deletion of genetic material
• Arrest of translation or transcription
• Activation of genes
• Generation of new plasmids
• resistance plasmids

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The U-tube experiment
after incubation, bacteria plated on minimal media
no prototrophs
demonstrated that direct cell to cell contact
was necessary
Figure 13.13
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RTF resistance transfer factor
a conjugative plasmid
R1 plasmid
sources of resistance genes are transposons
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Bacterial Conjugation

• transfer of DNA by direct cell to cell contact
• discovered 1946 by Lederberg and Tatum

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F x F Mating

• F donor
• contains F factor
• F recipient
• does not contain F factor
• F factor replicated by rolling-circle mechanism
  and duplicate is transferred
• recipients usually become F
• donor remains F

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F factor

• 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-".

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F x F mating

• 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.

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Hfr Conjugation

• Hfr strain
• donor having F factor integrated into its
  chromosome
• both plasmid genes and chromosomal genes are
  transferred

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Hfr x F mating
Figure 13.14b
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F? Conjugation
integrated F factor

• F? plasmid
• formed by incorrect excision from chromosome
• contains ? 1 genes from chromosome
• F? cell can transfer F? plasmid to recipient

chromosomal gene
Figure 13.15a
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F? x F mating
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Tra Y

• 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.

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Tra Genes

• 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)

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Conjugative Proteins

• 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

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DNA Transformation

• Uptake of naked DNA molecule from the environment
  and incorporation into recipient in a heritable
  form
• Competent cell
• capable of taking up DNA
• May be important route of genetic exchange in
  nature

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Streptococcus pneumoniae
nuclease nicks and degrades one strand
DNA binding protein
competence-specific protein
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Artificial transformation

• 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
• makes cells more permeable to DNA

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Transduction

• Transfer of bacterial genes by viruses
• Virulent bacteriophages
• reproduce using lytic life cycle
• Temperate bacteriophages
• reproduce using lysogenic life cycle

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Lysogenic Phage
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Lambda

• 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
  site-specific recombination reaction.

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Attachment site

• 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

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Recombination site

• 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.

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Integration

• 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.

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Enzymes and Recombination

• There are two major groups of enzymes that carry
  out site-specific 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.

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Excision of bacteriophages

• 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.

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Generalized Transduction

• 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

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Generalized transduction
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Specialized Transduction

• 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

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Specialized transduction
Figure 13.20
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Figure 13.20
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Mapping the Genome

• locating genes on an organisms chromosomes
• mapping bacterial genes accomplished using all
  three modes of gene transfer

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Hfr mapping

• 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

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Interrupted mating
Figure 13.22a
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Figure 13.22b
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E. coli genetic map

• gene locations expressed in minutes, reflecting
  time transferred
• made using numerous Hfr strains

Figure 13.23
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Transformation mapping

• used to establish gene linkage
• expressed as frequency of cotransformation
• if two genes close together, greater likelihood
  will be transferred on single DNA fragment

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Generalized transduction mapping

• 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

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Specialized transduction mapping

• provides distance of genes from viral genome
  integration sites
• viral genome integration sites must first be
  mapped by conjugation mapping techniques

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Recombination and Genome Mapping in Viruses

• 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

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Recombination mapping

• recombination frequency determined when cells
  infected simultaneously with two different viruses

Figure 13.24
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Physical maps

• heteroduplex maps
• genomes of two different viruses denatured, mixed
  and allowed to anneal
• regions that are not identical, do not reanneal
• allows for localization of mutant alleles

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Physical maps

• 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.