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Chapter 10 continued

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2. Joining the DNA fragments to a cloning vector (ex. plasmid or virus) with DNA ligase. ... plasmid pBR322 a good cloning vector? How does insertional ... – PowerPoint PPT presentation

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Title: Chapter 10 continued


1
Chapter 10 (continued)
  • Bacterial Genetics

2
Transformation vs. Transduction vs. Conjugation
3
Genetic Transformation
  • Genetic Transformation process by which free
    DNA is incorporated into a recipient cells and
    brings about genetic change.
  • A number of prok. are naturally transformable,
    including gram-pos. and gram-neg. Bacteria and
    some Archaea.
  • Only a small number of genes from one cell can be
    transferred to another by a single transformation
    event.

4
Competent Cells
  • Competent cells able to take up a molecule of
    DNA via transformation.
  • Only certain strains are competent and this
    ability is genetically determined.
  • Competence is regulated with special proteins
    playing a role in DNA uptake and processing.
  • ssDNA or dsDNA may taken up by cells, though it
    must be in ssDNA form to be incorporated into the
    genome by recombination.
  • Competent cells bind up to 1000X more DNA than
    noncompetent cells (dsDNA binds better to cells).
  • DNA fragments compete with each other for uptake.
  • While the max. frequency of transformation 20
    of the population, actual values 0.1-1.0.
  • Min. conc. of DNA yielding detectable
    transformants 0.00001 ?g/ml.

5
Integration of Transforming DNA
  • DNA is either taken up single-stranded or dsDNA
    is taken up and one strand is degraded ? ssDNA.
  • Next, the ssDNA associates with
    competence-specific protein that remains attached
    to the DNA to protect it from nuclease attack
    until it reaches the chromosome, where RecA takes
    over.
  • DNA is then integrated into the genome of the
    recipient by recombination.
  • During replication of this heteroduplex DNA, one
    parental and one recombinant DNA molecule are
    formed. On segregation at cell division, the
    recombinant DNA molecule is present in the
    transformed cell.

6
Transfection
  • Transfection Bacteria transformed by
    bacteriophage DNA instead of DNA from another
    bacterium.
  • Transfection by a lytic bacteriophage leads to
    normal virus production.

7
Artificially Induced Competence/Electroporation
  • Only a few bacteria exhibit natural
    transformation. Other bacteria can be made
    competent through artificially induced
    competence.
  • High conc. of cold Ca2 ions causes E. coli to
    become competent at low efficiency.
  • Electroporation a technique in which cells are
    exposed to pulsed electric fields to open small
    pores in their membranes. DNA present outside
    the cells can enter through these pores. This
    method works for a variety of prok. and euk.
    Plasmids can be transferred directly from one
    cell to another because DNA can exit as well as
    enter through these pores.

8
Transduction
  • Transduction DNA is transferred from cell to
    cell via viruses.
  • A variety of prok. can undergo transduction and a
    variety of phages can transduce.
  • 2 types of transduction ? virus ends up defective
    and homologous recomb. can occur in either case

    (1) generalized transduction
    host DNA derived from virtually any portion of
    the host genome becomes a part of the DNA of the
    mature virus particle in place of the virus
    genome.
    (2) specialized transduction
    occurs only in some temperate viruses DNA from
    a specific region of the host chromosome is
    integrated directly into the virus genome
    usually replacing some of the virus genes.

9
Generalized Transduction
10
Specialized Transduction
11
Phage Conversion
  • Phage conversion phenotypic alterations made in
    a lysogenized cell, can be acquisition of
    immunity to further infection by the same type of
    phage or can be some other change.
  • ex. toxin production in Corynebacterium
    diphtheriae.

12
Plasmids
  • Plasmids genetic elements that replicate
    independently of the host chromosome.
  • Thousands of different types of plasmids are
    known, almost all of which are dsDNA, most of
    which are supercoiled and circular, are vary in
    size from 1-1000 kbp.
  • Different plasmids are present in cells in a
    particular number of plasmid molecules per cell
    copy number, which can vary from 1-100.
  • Most gram-neg. plasmids replicate similar to the
    chrom., although some replicate unidirectionally.
    Most gram-pos. plasmids replicate by the rolling
    circle mechanism similar to a phage.

13
Plasmids (cont.)
  • Cells can contain different types of plasmids. A
    cell in which two plasmids cannot be maintained
    together are said to be incompatible.
  • Curing elimination of a plasmid from a cell.
    Curing can occur spontaneously or with the help
    of chemicals or electroporation.
  • Plasmids lack extracellular form.
  • The main mechanism of cell-to-cell plasmid
    transfer conjugation.
  • Plasmids that govern their own transfer by
    cell-to-cell contact conjugative.

14
Types of Plasmids
  • While all plasmids carry genes that ensure their
    own replication, some carry genes for
    conjugation, as well as other unique properties
    conferred upon the cell.
  • Resistance (R) plasmids confer resistance to
    antibiotics and other inhibitors of growth.
    These plasmids often transfer resistance to other
    cells via cell-to-cell contact, resulting in
    antibiotic resistant populations. R plasmids
    with genes for resistance to most antibiotics are
    known.
  • The following virulence factors of pathogenic
    bacteria can be encoded on plasmids

    (1) ability of
    microorganisms to attach and colonize specific
    sites in the host

    (2) formation of substances
    (ex. toxins, etc.) that cause damage to the host.
    What is this similar to that we just discussed?

15
Bacteriocins
  • Bacteriocins agents produced by bacteria that
    inhibit or kill closely related species or
    different strains of the same species. They are
    different from antibiotics, which have a wider
    spectrum of activity.
  • Bacteriocins are often plasmid-encoded.
  • Bacteriocins are named according to the organism
    that produces them.
  • They can interfere with another cells proton
    motive force and, thus, have practical uses such
    as food preservatives.

16
Conjugation and Chromosome Mobilization
  • What is bacterial conjugation also known as?
  • Conjugation is a plasmid-encoded mechanism, but
    can mobilize host chromosome as well.
  • The F plasmid of E. coli first confirmed the
    occurrence of conjugation.
  • Conjugation involves a donor cell containing a
    conjugative plasmid and a recipient cell, which
    does not. What are these cells also known as?
  • Sex pilus may be specified by the plasmid,
    allowing for specific pairing between donor and
    recipient. The pilus formed by the F plasmid is
    called the F pilus.

17
DNA Transfer During Conjugation
  • DNA synthesis is necessary for DNA transfer to
    occur.
  • Rolling circle replication model best explains
    DNA transfer during conjugation. This process is
    triggered by cell-to-cell contact.
  • At the end of the process, both donor and
    recipient possess plasmids and the recipient can
    become a donor, spreading the plasmids between
    populations like infectious agents.

18
Transfer of Plasmid DNA by Conjugation
19
Hfr (High Frequency of Recombination) Strains
  • F plasmid conjugative, can integrate into host
    chromosome ( episome), and can also mobilize
    chromosome transfer.
  • Cells with an unintegrated F plasmid F, while
    those having a chromosome-integrated F plasmid
    Hfr, and cells without and F plasmid F-.
  • Conjugation with Hfr donor ? transfer of host
    chromosome.
  • After transfer, an Hfr strain remains Hfr since
    it retains a copy of the F plasmid in the
    chromosome.
  • Note ori origin, ex. of replication or of
    transfer

20
F Plasmid
  • Cells having an F plasmid are able to synthesize
    and F pilus, mobilize DNA for transfer to another
    cell, and alter surface receptors so that the
    cell can no longer serve as a recipient.
  • The F plasmid can integrate into the host
    chromosome at sites called insertion sequences
    (IS) . Once integrated, the F plasmid no longer
    controls its own replication.
  • Usually, because of breakage of the DNA strand
    during transfer, only part of the donor
    chromosome is transferred. Although Hfr strains
    transmit chromosomal genes at high frequency,
    they usually do not convert F- strains to F or
    Hfr because the entire F plasmid is rarely
    transferred. However, F strains can convert F-
    strains to F because the entire F plasmid is
    transferred.

21
Interrupted Mating
  • Recombinants from conjugation can be selected
    for.
  • In an Hfr strain, the transfer of chromosomal
    genes will always occur in the same order from a
    fixed site on a given Hfr strain.
  • Interrupted mating interrupt mating pairs by
    agitation after a certain time that conjugation
    has occurred. Genes present closer to the origin
    enter the F- cell first.
  • This technique leads to genetic mapping since you
    can determine the order in which the genes occur
    by the order in which they are transferred.
    Genes at certain points can be referred to as
    positioned at so many minutes.
  • Genetic recombination is dependent on the
    occurrence of homologous recombination and is not
    a result of genetic transfer alone.
  • F cells in which the F plasmid has been
    excised from the chromosome and takes some
    chromosomal DNA with it.

22
Interrupted Mating Experiment
23
Other Conjugation Systems
  • Conjugative transposons can be transferred from
    the chromosome of a donor to a recipient and can
    mobilize other genetic elements.
  • Conjugative transposons are common to gram-pos.
    cells.

24
Complementation
  • Complementation is used to determine whether or
    not two mutations are in the same gene by
    restoring function of a gene by complementing the
    defective (mutant) gene with a normal (nonmutant)
    copy of that gene. Homologous recombination can
    restore gene function (unless both of the
    mutations include changes in exactly the same
    base pairs) but cannot reveal whether or not the
    mutations were in the same gene.
  • The two mutations are said to complement each
    other.
  • Bacterial gene transfer must be done in order to
    conduct this test.
  • Complementation does not involve recombination.
  • Cistron gene two mutations in the same
    cistron cannot complement each other.
  • In diploid organisms Cis 2 mutations from the
    same parent, Trans 2 mutations from different
    parent.

25
Complementation (cont.)
26
Transposons and Insertion Sequences
  • Some genes are capable of moving under certain
    conditions. The process by which a gene moves
    from one place to another in the genome
    transposition.
  • Transposition is relatively rare.
  • Not all genes are capable of transposition.
    Transposition of genes is linked to the presence
    of special genetic elements called transposable
    elements.
  • There are 3 types of transposable elements in
    Bacteria (1)
    insertion sequences

    (2) transposons

    (3) some special viruses (ex.
    Mu)
  • Transposable elements have 2 features in common
    (1)
    transposase enzyme necessary for transposition
    (2) inverted
    terminal repeats and the ends of their DNA.

27
Mechanisms of Transposition
  • Two mechanisms of transposition
    (1) Conservative
    the transposable element is excised from one
    location in the chromosome and becomes reinserted
    at a second location. The copy number of a
    conservative transposon remains at one. Direct
    repeats are formed in the target site at the ends
    of the transposon.
    (2)
    Replicative (ex. bacteriophage Mu) transposons
    are duplicated and a new copy is inserted at
    another location. A composition structure
    called a cointegrate is formed.
  • Transposition is essentially a recombination
    event, but one that does not occur between
    homologous sequences or use the general
    recombination system of the cell. It is called
    site-specific recombination and involves
    transposase instead of RecA.

28
Mechanisms of Transposition (cont.)
29
Mutagenesis with Transposable Elements
  • If the insertion site for a transposable element
    is within a gene, insertion of the transposon
    will result in loss of linear continuity of the
    gene, leading to mutation transposon
    mutagenesis means of creating mutants
    throughout the chromosome.

30
Integrons
  • Integrons genetic elements that can capture and
    express genes from other sources.
  • Integrons code for integrase, which catalyzes a
    type of site-specific recombination.
  • Integrase can integrate gene cassettes and a
    promoter that can then express the newly
    integrated gene cassette.
  • The genes in the gene cassette that are captured
    are not captured randomly, but have specific DNA
    sequences recognized by the integrase and genes
    that are not expressed until they become part of
    an integron.

31
Restriction Enzymes
  • Protect prok. from foreign DNA, ex. viruses.
  • Restriction enzymes recognize certain sequences
    of DNA and cut the DNA.
  • Palindrome sequence of bases that reads the
    same when read from either right or left.
    Palindromes are often the target of REs.
  • Introduce double stranded breaks.
  • In a random DNA molecule, one would expect any
    4-bp sequence to occur once every 256 bps based
    on the probability of 1/4 X 1/4 x 14 x 1/4.
  • A 6 bp sequence would appear every 4096 bps in
    random DNA and a 8 bp sequence would appear once
    every 1000 bps, so NotI cuts the E. coli genome
    (4600 bps) 21x, therefor the recognition sequence
    for NotI occurs more often than predicted.
  • There are over 2000 REs known with over 200
    specificities.

32
Protection from Restriction
  • Cells protect their own DNA from their REs by
    methods such as methylation of their own
    sequences that would be targeted by their REs.

33
RE Analysis of DNA
  • RE analysis is done by gel electrophoresis -
    whats the procedure for this?
  • Can be used to generate a physical map of DNA.
  • Nucleic acids can be purified from gels and used
    to transform cells or for nucleic acid
    hybridization as nucleic acid probes to find
    similar sequences from different genetic elements
    Southern blot (RNA hybridization Northern
    blot, protein hybridization Western blot).

34
Sequencing and Synthesizing DNA
  • 2 procedures (1) Maxim and Gilbert method (2)
    Sanger dideoxy method
  • Both methods generate DNA fragments that end at
    each of the four bases (G, A, T, C) and that are
    radioactive.
  • The fragments are subjected to gel
    electrophoresis in which 4 sample lanes are
    featured, one for each base.
  • Maxim-Gilbert method used chemicals that break
    the DNA preferentially at each of the four
    nucleotides.
  • Sanger dideoxy method sequence is determined by
    making a copy of the ssDNA using DNA pol., which
    used dNTPs as substrates, adding them to a
    primer. The dNTPs feature a dideoxy sugar analog
    that prevent lengthening of the chain and acts as
    a specific chain-termination reagent. Fragments
    of variable length are obtained. Either the
    dNTPs or primer are radioactive. This method can
    be used to sequence RNA as well.
  • Sequencing by the Sanger method is now automated
    and fluorescent labels have replaced radioactive
    ones.

35
Molecular Cloning
  • Gene cloning
  • Purpose isolate large quantities of specific
    genes or chromosomal fragments in pure form.
  • Basic strategy move the desired gene or region
    from a large, complex genome to a small, simple
    one.
  • Tools used restriction enzymes, DNA ligase,
    synthetic DNA (see 2 below).
  • Steps
  • 1. Isolation and fragmentation of the source DNA.
  • 2. Joining the DNA fragments to a cloning vector
    (ex. plasmid or virus) with DNA ligase. Blunt or
    sticky ends may be created on the ends of the
    source and/or vector DNA - what does this mean
    and how do you deal with each?
  • 3. Introduction and maintenance in a host
    organism. What types of organisms are used as
    host organisms (what are their characteristics)?

36
Molecular Cloning (cont.)
  • What makes plasmids good cloning vectors?
  • What is plasmid pBR322 a good cloning vector?
  • How does insertional inactivation work?

37
Polymerase Chain Reaction (PCR)
  • PCR requires that the nucleotide sequence of a
    portion of the desired gene be known because
    short oligonucleotide (- what does this mean?)
    primers complementary to sequences in the gene or
    genes of interest must be available for PCR to
    work.
  • Steps
  • 1. Two oligonucleotide primers flanking the
    target DNA are made (how?) and added to excess to
    heat-denatured target DNA.
  • 2. As the mixture cools, the target strands
    anneal mostly to a primer, which are in excess,
    and not to each other.
  • 3. DNA pol. then extends the primers using target
    strands as template.
  • 4. After an appropriate incubation period, the
    mixture is heated again to separate the strands.
    The mixture is then cooled to allow the primers
    to hybridize with complementary regions of newly
    synthesized DNA, and the whole process is
    repeated.

38
Polymerase Chain Reaction (PCR)
39
PCR (cont.)
  • Taq pol. is stable at 95C and is unaffected by
    the denaturation step. However, it has no
    proofreading activity.
  • Pfu pol. is stable at 100C and has proofreading
    activity, and is therefore more accurate.
  • PCR is often conducted in automated thermocycling
    machines.
  • It can be used to amplify very small quantities
    of DNA present in a sample.
  • It is not necessary for the organism to be grown
    in the lab, so it is important for environmental
    studies.
  • It can also be used for DNA fingerprinting, a
    powerful forensic tool permitting ID of
    individuals (crime scene/suspects) or
    relationships between individuals (paternity
    testing).

40
In Vitro and Site-Directed Mutagenesis
  • In Vitro in glass, i.e. in the lab external
    to the organism, as opposed to in vivo in the
    living organism. In other words, you can remove
    genes from and organism, manipulate them,
    engineer in mutations and put them back into an
    organism.
  • Site-directed mutagenesis Mutations can be
    introduced at precisely determined sites on
    genes.
  • Mutagenesis studies are often done on at the gene
    level to make amino acid changes to study protein
    structure.
  • Cassette mutagenesis segments of DNA can be
    manipulated in which synthetic fragments of DNA
    have replaced the wild-type sequence. These
    cassettes can be used for insertion inactivation,
    causing gene disruption. How is this done?

41
The Bacterial Chromosome
  • The entire genome of E. coli K-12 has been
    sequenced and has been found to be 4,639,221 bp
    with 4288 open reading frames, corresponding to
    88 of the genome (what are these and what is the
    rest of the DNA used for?).
  • The map distances on this genome are given in
    minutes of transfer, in which 0 time origin of
    transfer and 100 min. time the whole chromosome
    takes to be transferred from an Hfr strain to an
    F- strain.

42
The Bacterial Chromosome
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