Download Chapter 14 Transposons, Plasmids, and Bacteriophage

Chapter 14 Transposons,
Plasmids, and Bacteriophage
⁄ In this chapter you will learn
1. About transposable elements: how they
relocate, and their effects on gene expression
2. About the basic properties of plasmids, how
they replicate, and how they may be
transferred from cell to cell
3. About the life cycles of diverse
bacteriophages
Transposable Elements
Their Discovery Surprised
Molecular Biologists
1
Transposable Elements
⁄ These DNA sequences have unique
ability to move as a discrete unit from
one position in genome to another
– This ability to relocate is called transposition
– elements themselves are called transposable
elements (Tn elements)
– In bacteria are simply called transposons
Table 14-3 Properties of Several
E. coli Insertion Element
Element
Number of copies and location
Size in base-pairs
IS1
5-8, chromosome
IS2
5, chromosome; 1 in F
1327
IS3
5, chromosome; 2 in F
~1400
IS4
1 or 2, chromosome
~1400
IS5
Unknown
1250
gl
1 or more, chromosome; 1 in F
5700
768
2
What properties of IS sequences are
responsible for their remarkable ability to
transpose?
⁄ All elements contain a
terminally inverted
sequence
– IS1 has 23 bp terminal
repeat sequences; IS2
has a 41 bp repeat.
– Essential
Figure 14-7 An example of a
terminal inverted repeat. The
arrows indicate the inverted basesequences. Note that the sequences
AGTC and CTGA are not in the same
strand.
All IS elements are capable of encoding a special
DNA binding protein called a transposase
Direct repeat of
target sequence is a
consequence of a
staggered cut in the
target DNA made
by transposase
Figure 14-8 How
target sequence
might be duplicated
by formation of a
staggered cut at the
target sequence.
3
Complex Transposons
⁄ Consist of a gene or multiple genes, usually
encoding resistance to an antibiotic,
sandwiched between two IS elements
– Tn9: chloramphenicol; Tn10: tetracycline
n
Both IS
elements
flanking the
resistance
gene can be
capable of
independent
transposition.
More Complex
Transposons Exist
⁄ Between inverted repeats are three genes
– bla gene: b-lactamase that confers resistance
to ampicillin.
– tnpA gene: a transposase that specifically
recognizes inverted repeats
– tnpR gene: a bifunctional protein (resolvase)
• A specific DNA-binding protein that recognizes a
short DNA sequence called internal resolution
site (IRS).
4
More Complex
Transposons Exist
Consevative Transposition
⁄ It involves no replication of original element,
except for small amount needed to create
direct repeats flanking element at its new
location
– Excision of Tn element through cleavages at its
inverted termini by transposase
– Followed by insertion of the element at a new site
– Tn9, Tn10
5
Replicative Transposition
⁄Cointegrate form
– fusion of donor & target
replicons mediated by transposase
– resolved into two separate DNA
molecules, each containing a
copy of the transposon
– not by transposase but by
resolvase (product of tapR gene)
– Recombination step (resolution)
occurs at IRS sites in paired
copies in cointegrate called res
sites
Transposable Elements in
eukaryotes
6
First detected in Barbara
McClintock's studies of maize in
1940s
⁄ The best
characterized maize
Tn element is a 4.5
Kb element called
Ac (for activator)
⁄ defective versions of
Ac are called Ds (for
dissociation)
Ac/Ds cause mottled kernels
of corn
If an Ac element is also present in such a cell, it allows the Ds
element to jump out again & result is that the original cell
divides to form a mixture of purple & white cells
7
Transposable Elements in
eukaryotes
⁄ They are probably present in all
genomes
– Yeast, Drosophila melanogaster, and humans..
⁄ Retroviruses Infect Eukaryotic Cells and
Insert in Host Genome
⁄ Retrotransposons Make Up a Large
Proportion of Human Genome
The “Changing Genome”
Concept
⁄ The sequence or arrangement of
nucleotides in genes is constantly
evolving, either by random mutation,
by occasional recombination events,
or from the action of transposable
elements.
8
Plasmids
Plasmids
⁄ Most plasmids are circular, double-stranded
DNA molecules
– Isolated as supercoiled molecules
– From bacteria, yeast protozoa, & plants
⁄ Certain bacterial plasmids have ability to
transfer themselves from one cell to another
– Spreading through a population
– Play in bacterial evolution
⁄ Plasmids span a wide range of sizes
– Several Kb to > 500 Kb
⁄ They exist in characteristic copy numbers per
cell
9
Table 14-1 Features of Selected plasmids of
E. coli
Plasmid Size Copy
Conjugative Other Phenotype
(Kb) Number
ColE1
6.6
10-20
No
Colicin production and
immunity
F
95
1-2
Yes
E. coli sex factor
R100
89
1-2
Yes
Antibiotic -resistance
genes
P1
90
1-2
No
R6K
40
10-20
Yes
Plasmid form is
prophage; produces viral
particles
Antibiotic -resistance
genes
Plasmid-Borne Genes
⁄ Fertility or ‘F’ plasmids
– tra genes, able to promote conjugal transfer
⁄ Resistance or ‘R’ plasmids
– resistance to antibiotics or heavy metals
⁄ Col plasmids
– Colicins - proteins that kill other bacteria
⁄ Degradative plasmids
– metabolize unusual molecules such as toluene &
salicylic acid.
⁄ Virulence plasmids
– Pathogenicity
10
Table 14-2 Plasmid-encoded proteins
Protein
Function
Colicin
Secreted protein, kills bacteria lacking
plasmid that encodes colicin-immunity
protein
Enterotoxin
Secreted protein, alters ion balance of
eukaryotic cells. Responsible for water loss
from cells
Plasmid Transfer: conjugation
Figure 14-1 Electron micrograph
of two E. coli cells during
conjugation. The small cell is an Fcell; the large cell contains F’ Lac.
Pilus: a conjugation bridge
⁄ Conjugative plasmids carry a large block of transfer genes
responsible for specialized cell structures & enzymes
required to physically move plasmid genome from a donor
cell to a recipient
⁄ Such plasmids are said to be
self-transmissible
11
Prototype conjugative plasmid of E.
coli is F plasmid (sex factor)
⁄ A large circular
plasmid of ~95 Kb
present in 1-2 copies
per cell
⁄ Cells lacking F plasmid
(F-, females); cells
having an F plasmid
(F+, males)
Figure 14-2 A map of the sex plasmid F. The single capital letters refer to the
midpoints of the locations of the corresponding tra genes. The insertion sequences gd
(Tn1000), IS2, and IS3 are shown in red. The red arrow (oriT) shows the location of
the origin for transfer replication as well as the direction of transfer. oriV shows the
location of the origin for vegetative (nontransfer) replication.
1.
Cell-cell contact.
2.
Nicking of F plasmid DNA at oriT.
Attachment of protein(s) to free 5’
terminus.
3.
Initiation of DNA synthesis from free 3’OH in dornor cell to generate rolling
circle replication intermediate. Entry of
5’ end of F into female.
4.
Continued replication in donor cell.
Initiation of DNA replication in
recipient cell.
5.
Completion of replication in donor cell.
Cleavage of transferred strand from
replication intermediate. Formation of
two complete circular F DNA molecules.
6.
Sealing of gaps and supercoiling to
generate two F+ cells
12
F plasmid Can Transfer Chromosomal Genes
by Recombination with the Host Genome
1.
Formation of mating pair
2.
Hfr chromosome is nicked in
integrated F DNA sequence
3.
Transfer of donor
chromosome, starting with F
sequence, occurs into female
cell. Replacement DNA
synthesis occurs in donor Hfr.
4.
Mating pairs usually separate
before complete transfer
occurs, in case after c+ gene has
been transferred but before d
gene has entered recipient cell.
F plasmid Can Transfer Chromosomal Genes
by Recombination with the Host Genome
5. RecA-mediated
recombination occurs
between transferred DNA
& host chromosome
6. Following chromosome
replication, two cell types
exist
one like original Fa second in which b and c
genes have been replaced
13
Hfr Transfer Can Be Used
to Investigate Gene Location
Figure 14-5 The course of entry of different genetic markers
into an F- recipient. Marker 1 is transferred early; marker 4 is
transferred later.
⁄ Interrupted mating
Plasmid DNA Replication
⁄ Most E. coli plasmids use DNA polymerase III
as central replication enzyme
– An important exception is ColE1 plasmid family
– In which DNA polymerase I plays the key replication
role
⁄ Rep protein, it plays a key role in initiation of
plasmid replication.
– Plasmid copy number is related to intracellular level of
Rep protein.
– Function of Rep protein is to recognize specific short
DNA sequences at plasmid ori
– Replisome
14
Plasmid Copy Number
⁄ A ratio of number of plasmid molecules
to number of chromosomal copies per
cell
– Naturally occurring plasmids exist at a wide
range of copy numbers
– Sex factor F or prophage of E. coli cirus P1,
exist at copy numbers of 1-2
– The ColE1 family exist at copy numbers of 2040
Bacteriophage
15
The three basic structures
Figure 14-10 Two-dimensional view of three basic phage
structure. The nucleic acid is shown in blue
Properties of nucleic acid of
several phage types
16
Stages in the Lytic Life
Cycle of a Typical Phage
Injection sequence of a
tailed phage
⁄ In the injection stage, the tail
sheath contracts and drives a
core protein tube through the
cell wall like a hypodermic
syringe
17
life cycle of E. coli phage T4
(times in minutes at 37˚C)
⁄t=0
⁄t<1
⁄ t = 2-3
⁄t=5
⁄
⁄
⁄
⁄
t=9
t = 12
t = 13
t = 25
Phage adsorbs to bacterial cell wall.
Injection of phage DNA
Synthesis of first phage mRNA begins
Synthesis of host DNA, RNA, & protein is
turned off
Degradation of bacterial DNA begins.
Phage DNA synthesis is initiated.
Synthesis of late mRNA begins
Completed heads & tails appear
First complete phage particle appears
Lysis of bacteria-, release of about 300
progeny phage
Specific Phage
E. coli phage T4,
E. coli phage M13,
E. coli phage T7
E. coli phage l
18
E. coli phage T4
⁄ Genome: 166,000 bp, ~200 average-size
genes
– Less dependent on host functions
– More complex life cycle
⁄ T4 DNA replication proceeds normally
in thymine-requireing hosts in absence
of added thymine
– T4 encodes its own thymidylate synthetase
E. coli phage T4
⁄ T4 DNA contains a
modified cytosine
– 5-hydroxymethylcytosine
(HMC)
– It base pairs with guanosine
⁄ T4 also encodes nucleases
– Attack cytosine-containing
DNA
– Degrade host chromosome
19
T4 DNA are terminally
redundant
⁄ That is, a sequence of bases (about 1%) is
repeated at both ends
⁄ Although each phage contains one DNA
molecule
– The molecules differ from phage to phage
– Circularly permuted
Circularly permuted
20
Figure 14-16 Proposed
model for filling a T4 head
⁄ Cleavage & rearrangement of head proteins is
known to occur at several stages of the process
Headful mechanism
⁄ Figure 14-17
Origin of
circularly
permuted T4
DNA molecule.
Alternate units
are shown in
different colors
for clarity only
21
E. coli phage T7
⁄ DNA: MW = 26 x 106, no unusual bases
⁄ A terminal redundancy of 160 base pairs
– But it is not circular permuted
⁄ T7 has fewer genes than T4 (about 50)
T7 transcription occurs in
three temporal stages
22
E. coli phage M13
⁄ It is one of the
smallest known phage
⁄ Genome: only 10 genes
– Almost all of them are
essential
– A circular DNA
molecule [viral or (+)
strand]
– Only 6,407 nucleotides
long
Replication
of phage M13
23
Replication
of phage M13
E. coli phage l:
The lytic cycle
⁄ A temperate E. coli
phage
– A lytic cycle
• Progeny phage are
produced
– A lysogenic cycle
• A phage-DNA
molecule is inserted
into bacterial
chromosome
24
Gene arrangement of
phage l
⁄ Clustering &
transcription of
numbers of a cluster
from same DNA strand
– Allow l to regulate
transcription with a
small number of
regulatory sites
Figure 14-24 The
replication sequence of
phage l
⁄ Earliest stage of replication of l DNA is bidirectional q
mode (figure 7-4)
⁄ Progeny circles do not continue to replicate in this
way, but switch to the rolling circle mode, yielding a
long linear branch
25
Figure 14-25 The mode of
cutting l units from a
branch of a rolling circle
The units are cut sequentially from the free end by terminase
E. coli phage l:
The lysogenic life cycle
⁄ A lysogen is immune to infection by a phage of the type
that lysogenized the cell
⁄ Induction:
– Even after many cell generations, a lysogen can initiate a lytic
cycle
– prophage is excised as a circular DNA molecule & initiates lytic
growth
26
What determines whether a l
infection will be lytic or lysogenic?
⁄ cII protein
– A transcription activator that binds to two promoter
regions of genes that are required for lysogeny
⁄ High levels of cII favor lysogeny, while low
levels favor the lytic pathway
– Instability of cII protein is due to degradation by
proteases
– Under favorable growth conditions, proteases are
plentiful, cII protein is degraded, &, hence, lysogenic
cycle is not activated
– Under poor growth conditions, proteases are less
abundant & there is less degradation of cII, so those
promoters are activated & lysogeny is favored
Transposons, Plasmids, &
Bacteriophage in Genetic Engineering
Figure 14-29 Use of
various genetic elements
for recombinant DNA
procedures
27