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Transposons
※
Transposons are DNA elements that can hop, or transpose, from one
place in DNA to another. They are also called “jumping genes”.
They carry the enzyme, transposase responsible for transposition,
the movement by a transposon.
※ They are discovered by Barbara McClintock in the early 1950s.
※ The transposons now exist in all organisms on the earth, including human.
※ Transposons may offer a way of introducing genes from one bacterium into
the chromosome of another bacterium to which it has little DNA sequence
homology, so they obviously play an important role in evolution.
※ Transposition must be tightly regulated and occur only rarely;
otherwise, the cellular DNA would become riddled with the transposons,
which would have many deleterious effects. In fact, the frequency of
transposition varies from once in every 103 to once in every 108 cell division,
depending on the type of transposon. It is not higher than the chance of a
gene inactivated by other mutation. Almost half of human genome may be
the transposons.
※ Genome – The complete DNA sequence of an organism.
人類基因體計畫
1. 人類的染色體為 23 對，其一半即為構成人類的基因體，約含有 3 X 109 鹼基對
（bp）
，其大小約為大腸桿茵（4.2 X 106 bp）的 1,000 倍，這是否表示人類的
基因體所含的基因數目為大腸桿茵的 1,000 倍（大腸桿茵有 2,000 個基因）。
答案是否定的，人類基因體計畫的完成顯示人類的基大約有 35,000 個。所以
人類基因體含有大量的〞廢物 DNA（junk DNA）〞
，約佔人類總 DNA 的 97％，
這些 DNA 包括基因中的隱子，基因間的重覆序列及所謂的跳躍基因。
2. The largest of component of the human genome consists of transposons. Other
repetitive sequences include large duplications and simple repeats.
• Overview of transposition
Types of bacterial tansposons
1. Insertion sequence (IS) elements
(1) These transposons are usually only about 750 ~ 2000 bp long and encode
little more than the transposae that promote they transposition.
(2) Repeats at ends, usually inverted repeats.
(3) IS3 consists of two open reading frames (ORFA and ORFB).
(4) ORFB is shifted -1 relative to ORFA, but a programmed -1
(2) Fig.9.2 is the structure of the IS3 element, which contains
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frameshift causes the synthesis of a fusion protein, ORFAB,
which is the active transposase.
(5) Smaller protein made from ORFA when the frameshift does
not occur regulates transcription of transposase.
(6) The target site sequence that is duplicated on the insertion of IS3 is 3 bp
long (ex., ATT). The length of such direct repeats is characteristic of
each type of transposon.
Structure of the insertion sequence element IS3 and its related family member
1. The inverted repeats are shown as arrows, and the 3-bp target
sequence that is duplicated after transposition is boxed.
2. OFRA and OFRB encode the N terminus and C terminus of the
transposase, which are translated in different reading frames and are not
active by themselves.
3. A programmed -1 frameshift put both ORFA and ORFB in the same frame and
makes the active transposase. The C terminus of the IS3 transposase
contains the DDE motif characteristic of this type of transposase.
2. Composite transposons
(1) A larger transposon: Two IS elements of the same type bracket other genes,
usually the antibiotic resistant gene(s).
(2) Transposition
* Each IS element of the same transposon can transpose independently as
long as the transposase acts on both of its ends.
* Two IS elements are often not completely autonomous, because the
active transposase of one IS element can act on the outside ends to
promote transposition of the composite transposon when the
transposase of the other element is inactive due to a mutation.
i. Outside-end transposition
When a transposase acts on the inverted repeats at the farthest ends of
a composite transposon, the two IS elements transpose as a unit along
with the genes between them.
ii. Inside-end transposition
A transposase encoded by one IS element can also act on the inside
ends of both IS elements.
Structures of some composite transposons
1. The active transposase gene is in one of the two IS elements.
2. The IS elements can be in either the same or opposite
orientation (arrows).
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• Two IS elements can transpose any DNA between them
• Either the outside or inside ends of the IS elements in a composite
transposon can be used for transposition
(a) Transposition with the outside ends of IS10 element would
move Tn10, with the tetracycline resistance gene (Tetr) to another
DNA.
• Either the outside or inside ends of the IS elements in a composite
transposon can be used for transposition
(a) Transposition with
the outside ends of
IS10 element would
move Tn10, with the
tetracycline
resistance gene
(Tetr) to another
DNA.
• Rearrangements of DNA caused by composite transposons through
inside-ends transposition - neighboring sequences between the original site
of insertion of the transposon and the site into which it is trying to
transpose
will be either deleted or inverted. (B, C, D)
4. Methods have been developed to select tet-sensitive
derivatives of E. coli haerboring the Tn10 transposon. Most of
these tet-sensitive derivatives have deletions or inversions of
DNA next to the site of insertion of Tn10 element.
5. Inside-end transposition is presumably responsible for most of
the often-observed instability of DNA (rearrangement) caused
by composite transposon.
6. Some composite transposons have mechanisms to avoid
inside-end transposition. Ex., adenines of inside-ends of Tn5
are methylated so that they are recognized less well by the
transposase.
7. Assembly of plasmids by IS elements
- Many of the resistance gene on plasmids are bracketed by the
same IS element. Apparently, the plasmid was assembled in
nature by resistance genes hopping onto the plasmid from
some other DNA via the bracketing IS elements.
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III. Mechanisms of transposition
IIIa.
A molecular model for transposition of Tn3 (A replicative transposition)
1. Breaks are made in the target DNA and at the ends of the
transposon, respectively (1 and 2).
2. The 3’ OH ends of the transposon (dots) are ligated to 5’
PO4 ends of the target DNA (3).
3. The free 3’ ends of the target DNA prime replication in both
directions over the transposon to form the cointegrate (4).
4. The cointegrate is resolved by recombination promoted by
the resolvase TnpR at the res sites (5)
5. Resolution of the cointegrate give rise to two copies of the
transposons, one at the former (or donor) site and a new
one at the target site.
(The A and B in the target DNA illustrate how the target
DNA is reversed in the step 3 for ease of drawing.)
6. The transposase cuts the target and donor DNAs and
promotes ligation of the ends.
7. The resolvase specifically promotes recombination
between the res elements in cointegrate.
8. Mu phage replicate itself and insert itself around the
chromosome of its bacterial host by a mechanism similar
to Tn3.
(1) It does not resolve the cointegrate and soon the
chromosome becomes riddled with Mu genome.
(2) These genomes are packaged directly from chromosomal DNA into
the phage head, discarding the
bacterial chromosomal DNA between the inserted Mu
genomes.
IIIb. Transposition by Tn10 and Tn5
• Transposition by a cut-and-paste mechanism (also known as conservative
mechanism or nonreplicative transposition)
• The transposon is moved from one place and inserted into another place.
• Transposon produces a short duplication of target DNA at the ends of the
transposon.
• Donor DNA probably leaves break and is consequently degraded.
• There is no cointegrate formation as it does in the replicative mechanism.
Details of the mechanism of transposition by Tn5
1. Single copies of the transposae (TnP) bind to each end of the transposon,
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and then bind to each other, bring the two ends of transposon together
(synapsis).
2. Transposase bound at one end cuts the DNA at the other end and vice versa
to leave 3’ OH ends at each end of transposon.
3. These activated 3’ OH ends attack the phosphodiester bond on the other
strand, forming 3’-5’ phosphodiester hairpins. This cuts the transposon out
of the donor DNA.
4. When the transposase binds to the target DNA, it cuts the two hairpin ends
again and the 3’ OH ends attack phosphodiester bonds 9 bp apart in the
target DNA, cutting them, and the 5’ phosphate ends in the target DNA are
joined to the 3’ OH ends in the transposon, inserting the transposon into
the target DNA.
5 .The 9-bp single-stranded gaps on each side of transposon are filled in by
DNA polypomerase to make the 9-bp repeats in the target DNA.
Details of the mechanism of transposition by Tn7
• The cut and paste transposon Tn7 can be converted into a
replicative transposon by a single amino acid change in one
subunit of transposase.
• Different subunits of transposase make the cuts in the opposite
strands of DNA at the ends of transposon.
- TnsA cuts at the 5’ and, and TnsB cuts at 3’ end,
- They cut the donor DNA only in the presence of the target
DNA.
• If the TnsA subunit that makes the cut that leaves the 5’
hydroxyl end is altered by a mutation, transposase will cut only
the other strand, leaving a free 3’ OH like a replicative
transposase.
* Apparently, the transposases need only make the appropriate
cuts and joinings, and the replication apparatus of the cell does
the rest.
* The linear Tn7 transposon does not cut itself out of the donor DNA
unless the target DNA is already bound to the transposase, so the 5’ ends are
not left exposed for long.
The major difference between replicative and nonreplicative
transposition
1. Replicative transposase cuts only one strand at the junction.
2. Nonreplicative transposase makes cuts in both strands in the junction.
The similarity between replicative and nonreplicative transposition
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1. The cut 5’ ends of the target DNA are joined to the free 3’ ends of the
transposon.
2. The free 3’ ends of target DNA are used as primers for replication that
proceeds until a free 5’ end in the donor DNA is reached (The only different
is whether the replication has to proceed over the entire transposon
(replicative ) or only over the short region that is duplicated (cut and paste).
IV. General properties of transposons
• Target specificity
1 . No transposable element inserts completely randomly into
target DNA: Target specificity of some transposons are
relaxed and some are stringent.
2. Tn7 transposes with a high frequency into only one site in E.
coli DNA, called attTn7, just downstream of the glmS gene.
(1) The transposition machinery consists of five proteins:
i. TnsA and TnsB – make up the transposase that cuts and
joins the DNA strands.
ii. Other proteins play ancillary roles:
(i) TnsD – may direct the Tn7 to the target sequence,
attTn7. It may induce changes representative of
triple-stranded structures in the attTn7 site.
(ii) TnsC – (i) event directs TnsC to stimulate transposition into
the site.
(iii) TnsE – In the absence of TnsD, TnsE stimulates transposition into
other site on chromosome. This transposition is
inefficient but random.
(2) The glmS gene is highly conserved.
i. The product of glmS performs an important step in cell wall
biosynthesis.
ii. The insertion site of Tn7 is downstream the gene, and has no effect
on cell only its transcription termination site.
• Effects on genes adjacent to the insertion site – could be negative or
positive (Tn5 and Tn10 contain promoter near their termini)
• Regulation of transposition – transposition of most transposons occurs
rarely because they self- regulate their transposition.
The regulatory mechanisms differ greatly:
1. Tn3 – The TnpR protein represses the transcription of the
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transposase gene (Tnp).
2. Tn10 – transposition occurs primarily just after a replication fork has
passed through the element.
(1) Newly replicated E. coli DNA is hemimethylated at GATC sites, and it not
only activates the transposase promoter but also increases the
activity of the transposon ends.
(2) The translation of transposase is also repressed by an
antisense RNA.
3. Tn5 - Using a truncated transposase version to inhibit the
active one.
(1) Two similar IS50 elements flank the antibiotic resistance genes.
(2) An N terminally truncated Tnp (transposase) inhibit the active
one.
(3) Dam methylation of the inside ends (IEs) of the IS50 prevents
the transposase from cutting IEs and transposing the individual IS50
elements.
• Target immunity – Some transposons prefer not to hop close in the DNA to
another transposon of the same type. Immunity can extend over 100,000
bp of DNA.
(1) If two transposons were to insert close to each other, would
cause large deletions and often lead to the death of the cell.
Also, the presence of two transposons close to each other
can cause instability in chromosome.
(2) Only Mu, Tn3 and Tn7 families of transposons are known to
exhibit target immunity.
i. MuB protein seems to be indirectly resposible for the immunity.
ii. The binding of MuB to a DNA make it a target for the MuA
transposase, which then promotes transposon into DNA.
iii. The binding of MuA then cause MuB to dissociate from DNA.
iv. Once a transposon has inserted, a copy of MuA may remain bound to
the end of the inserted transposon, and prevent the binding of other
MUB to the same target DNA and other transposition into that DNA.
v. A similar mechanism may explain target site immunity by Tn7, and
the resposible proteins are TnsB and TnsC.
V.Transposon mutagenesis
• Transposons are useful for mutagenesis should have the following
properties:
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1. It should transpose at a fairly high frequency.
2. It should not be very selective in its target sequence.
3. It should carry an easily selectable gene, such as
one for resistance to an antibiotic.
4. It should have a broad host range for transposition if it is to be used in
several different kinds of bacteria.
• Transposon Tn5, for example in many types of G – bacteria.
• There are no equally universal methods for G + bacteris.
(A) A standard protocol for transposon mutagenesis of
G- bacteria:
1. A suicide ColE1-derived plasmid contains a mob site and
transposon Tn5 .
2. The relaxase of this suicide plasmid recognizes the coupling protein
of promiscuous plasmid RP4.
3. This suicide plasmid is mobilized into the bacterium by the
products of the RP4 transfer genes, which are inserted in the
chromosome.
4. Tn5 hops into the chromosome of the recipient cell, and the
ColE1 plasmid is lost because it can not replicate.
(B) Random transposon mutagenesis of a plasmid
1. Transposon Tn5 is introduced into cells on a suicide vector.
2a. The culture is incubated, allowing the Tn5 time to hop, either
into the chromosome (large circle) or into a plasmid (small circle).
2b. Plating on kanamycin-containing medium results in the
selection of cells in which a transposition has occurred.
3. Plasmid is prepared from Kanr cells and used to transform
Kans cells.
4. Selection for Kanr allows the identification of cells that has
acquired a Tn5-carrying plasmid.
• Cloning genes mutated with a transposn insertion
1. A transposon used for mutagenesis of a chromosome contains a plasmid
origin of replication (ori).
2. The chromosome is cut with a restriction enzyme, ex., EcoRI which no cut
in transposon, and religated.
3. Transform E. coli with ligated mix, the resulting plasmid in the Ampr
transformants will contain the chromosomal sequences that flanked the
transposon .
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Transformation
• DNA can be exchanged among bacteria in three ways:
1. Conjugation – a plasmid or other self-transmissible DNA element
transfers it self and sometimes other DNA into other bacterial cell.
2. Transduction – a phage carries DNA from one bacterium to another.
3. Transformation – cells take up free DNA directly from their envirnment.
• Naturally transformable bacterium
(Most types of cells cannot take up DNA efficiently unless
they have been exposed to special chemical or electrical treatments to
make them more permeable.)
1. Naturally transformable bacterium (or naturally competent bacterium) –
They can take up DNA from the environment without requiring special
treatment.
2. About 40 species have been found to be naturally competent or
transformable.
3. Bacillus subtilis, Streptococcus pneumoniae, Haemophilus influenzae,
Neisseria gonorrhoeae, Helicobacter pylori, Acinetobacter baylyi, and
some species of marine cyanobacteria.
• Artificially induced competence
Bacteria can be sometimes be made competent by certain chemical
treatments or DNA can be forced into bacteria by a strong electric field in a
process called electroporation.
1. Treatment with calcium ions.
(1) Chemically induced transformation is usually inefficient, and only a
small percentage of the cells are ever trnasformed.
(2) Accordingly, the cells must be plated under conditions selective for the
transformed cells.
(3) Therefore, the DNA used for the transformation should contain a
selectable gene such as one encoding resistance to an antibiotic.
2. Electroporation
(1) The bacteria are mixed with DNA and briefly exposed to a strong
electric field.
(2) The bacteria must first be washed extensively in buffer with very low
ionic strength such as distilled water. The buffer usually also
contains a nonionic solute such as glycerol to prevent osmotic
shock.
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(3) The brief electric field across the cellular membranes might create
artificial pore of H2O lined by phospholipid head groups.
pass through these temporary hydrophilic pores.
(4) Electroporation requires specialized equipment.
DNA can
Discovery of transformation
1. In 1928, Fred Griffith found that one form of the pathogenic pneumococci
(now called Streptococcus pneumoniae) could be mysteriously
“transformed” into another form.
2. Griffith made a conclusion that the dead pathogenic bacteria gave off a
“transforming principle” that changed the live nonpathogenic
rough-colony-forming bacteria into the pathogenic smooth-colony form.
3. Later, other researchers did an experiment in which they trnasformed
rough-colony-forming bacteria into the pathogenic smooth-colony form by
mixing the rough forms with extracts of the smooth forms in a test tube.
4. About 16 years later after Griffith did his experiment with mice, Oswald
Avery and his collaborators purified the “transforming principle” from
extracts of smooth-colony formers and showed that it is DNA. Avery and
his colleagues were first to demonstrate that DNA, and not protein or other
factors in the cell, is the hereditary material.
Competence
• The ability of some bacteria to take up naked DNA from their environment.
• It is genetically programmed. Generally, more than a dozen genes are
involved, encoding both regulatory and structural components.
• The general steps that occur in natural transformation differ somewhat in
Gram-negative and positive bacteria.
• The followings are two examples for G – and G + , respectively.
• The steps for DNA uptake
1. Binding of double-stranded DNA to the outer cell surface of bacterium.
2. Movement of DNA across the cell wall and outer membrane (no outer
membrane in G + bacterium).
3. Degradation of one of the DNA strands.
4. Translocation of the remaining single strand of DNA into cytoplasm of the
cell across inner membrane.
5. Once in the cell, the single-stranded transforming DNA might synthesize
the complementary strand and reestablish itself as a plasmid, stably
integrate into the chromosome, or degraded.
• While the DNA uptake system of G + and G – bacteria have
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features in common, they do seem to differ in certain important
respects.
1. There are many proteins involved in transformation in bacteria.
2. They are discovered on the basis of isolation of mutants that
are completely lacking in the ability to take up DNA.
3. The genes affected in the mutants were named com (for
competence defective).
(1) The com genes are organized into several operons.
(2) The products of these, including the comA and comK
operons, are involved in regulation of competence.
(3) Others, including the products of genes in the comE, comF,
and comG operons, become part of the competence
machinery in the membrane that takes DNA up into the
bacteria.
(4) The genes in these operons are given two letters, the first for
the operon and the second for the position of gene in the
operon, ex., comFA is the first gene of the comF operon.
• Steps in natural transformation
1. ComEA encoded by the first gene of the comE operon, binds directly
extracellular double-stranded DNA.
2. The comF genes encode proteins that translocate the DNA into the cell.
ComFA is an ATPase that may provide the energy for
translocation of DNA through the membrane (not shown).
3. ComEA, ComEC, and ComFA form a sort of ATP-binding
cassette (ABC) transporter, which transports DNA into the cell.
4. The genes in the comG operon encode proteins that might form a
“pseudopilus” which helps move DNA through the ComEC channel.
They might bind to extracellular DNA, perhaps acting through the
ComEA DNA-binding protein, and then retract, drawing the DNA into the
cell.
5. The comE, comF and comG operons are all under the transcrptional
control of ComK, a transcription factor that
is itself regulated by ComA.
6. Some of genes involved in the transformation process are not designated
as com, because such genes were first discovered on the basis of their
involvement in other processes.
(1) The nucA gene product makes double-strand breaks in extracellular
DNA. The free DNA ends become the substrates for the
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competence proteins.
(2) Other examples are single-stranded-DNA binding protein (SSB), and
RecA functions in the recombination of transforming DNA with
chromosome DNA.
• The lengths of single-stranded DNA incorporated into the recipient
chromosome are about 8.5 to 12 kb based on cotransformation of genetic
markers, and the incorporation takes only few minutes to be completed.
• The proteins in shaded boxes are analogous in G +
and G – bacteria. ComEC of B. subtilis is an ortholog of ComA protein of
Neisseria.
• The DNA is shown running through the cell wall alongside the pseudopilus
(ComG in B. subtilis; PilE in G – systems that are related to type II protein
secretion systems.)
• Natural transformation of Gram-positive bacteria
• The comF genes encode proteins that translocate the DNA into
the cell.
• ComEA, ComEC, and ComFA form a sort of ATP-binding cassette (ABC)
transporter.
• The genes in the comG operon encode proteins that might form
a “pseudopilus” which helps move DNA through the ComEC
channel, and the ComECs retract,drawing the DNA into the cell.
Natural transformation of Gram-negative bacteria
1. ComA protein of Neisseria is an ortholog of ComEC of B. subtilis.
2. The DNA is shown running through the cell wall alongside
the pseudopilus (ComG in B. subtilis; PilE in G – systems).
3. In most G – bacteria specific sequences are required for the
binding of DNA, so that these species usually take up DNA only
of the same species.
Regulation of competence in B. subtilis
It is achieved through a two-component regulatory system: a sensor protein
(ComP) and a response regulator (ComA) protein.
1. When the cell runs out of nutrients and the population reach a high density
registered by ComP.
2. ComP autophosphorylates itself.
3. ComP ~P transfer ~P to ComA.
4. ComA~P is an active transcriptional activator for several genes,
including some required for competence.
5. Eventually, another transcriptional activator, ComK is made.
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It is directly responsible for activating the transcription of other
com genes, including those that form the transformation
machinery.
How does the cell know that other B. subtilis cells are nearby and that it should
induce competence?
1. High cell density is signaled through small peptides, competence pheromones
that are excreted by the bacteria as they multiply.
2. Cells become competent only in the presence of high concentrations of these
peptides.
3. This is a phenomenon called quorum sensing. The small
molecules are known as including homoserine lactones that
signal cell density in some G – bacteria.
4. In B. subtilis, the major competence pheromone peptide is
called ComX and is cut out of a longer polypeptide, the product
of the comX gene.
5. The product of gene comQ which is immediately upstream of
comX is a protease that cut the longer polypeptide.
6. Once the peptide has been cut out of the longer molecule, it
binds to the ComP protein in the membrane and trigger its
autophosphorylation.
7. At best, only about 10% of B. subtilis cells ever become
competent, no matter how favorable the conditions or how high
the cell density. This has been called a bistable state and
seems to be determined somehow by autoregulation of the
ComK activator protein.
Regulation of competence development in B. subtilis by quorum sensing
A
1. ComP in the membrane senses a high concentration of the ComX peptide,
and phosphorylates itself by transferring a phosphate from ATP.
2. The phosphate is then transferred to ComA.
3. ComA activates the transcription of many genes including comK.
4. ComK is an activator of the com genes.
B
1. In another pathway, a peptide sometimes called CSF
(competence-stimulating factor) processed from the signal
sequence of another protein (PhrC) is imported into the cell by
the SpoOK oligopeptide permease.
2. CFS indirectly activates ComA~P by inactivating RapC.
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Relationship between competence, sporulation, and other cellular states
1. About the same time as B. subtilis reaches the stationary phase, some cells
acquire competence and some cells sporulate, forming the endospore.
2. Sporulation allows a bacterium to enter a dormant state and survive adverse
conditions, such as starvation, irradiation and heat.
3. To coordinate sporulation and competence, B. subtilis cells may produce
other competence peptide.
(1) There are at least two such peptides that regulate ComA indirectly by
inhibiting proteins, Rap proteins, which bind to the C-terminal
DNA-binding domain of ComA~P and prevent it from binding to DNA
and activating transcription.
(2) These peptides (CSF) are processed from the signal sequences of longer
polypeptides, the products of the phr genes, and are transported into
cell by the oligopeptide permease, SpoOK.
(3) The spoOK gene is an example of a regulatory gene
that is required for sporulation and also for the development of
competence.
Three questions for natural transformation
A. How efficient is DNA uptake?
- Donor DNA is radioactively labeled by growing the cells in
medium containing 32P.
- The radioactive DNA is then extracted and mixed with competent cells.
- The mixture is treated with DNase at various times.
- Any DNA that is not degraded and survives intact must have been taken up
by the cells, where it is protected from the DNase.
- Collect cells on filter and count the radioactivity. Degraded DNA will pass
through filter.
- The radioactivity on the filter is compared with the total
radioactivity of the DNA that was added to the cell.
- This kind of experiment shows that some competent bacteria take up DNA
very efficiently.
B. Can only DNA of the same species enter a given cell?
- The same experiment demonstrates that some types of
bacteria take up DNA from only their own species (ex.,
Neisseria gonorrhoeae and Haemophilus influenzae) whereas
others (B. subtilis) can take up DNA from any source.
- Bacteria that preferentially take up the DNA of their own
species do so because their DNA contains specific uptake
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sequences.
Transformation in Streptococcus pneumoniae
1. Competence-stimulating peptide accumulates as the cells
reach a high density.
2. Double-stranded DNA binds to the cell, and one strand is
degraded.
Transformation in Haemophilus influenzae
3. The basic transformation scheme may differ among different
types of naturally competent bacteria.
4. In H. influenzae, the double-stranded DNA may first take up in
subcellular compartments called transformsomes.
C. Are both of the DNA strands taken up and incorporated into the cellular DNA?
- Experiments have shown that only double-stranded DNA can bind to
specific receptors on the cell surface, i.e., single-stranded DNA can not
transform cells and yield recombinant types.
- However, the transforming DNA enters a “eclipse” period for a short time
after it is added to competent cells, as expected if it enters the cell in a
single-stranded state.
- The following is the design for experiment:
Whether both of the DNA strands taken up and incorporated into the cellular
DNA?
As shown in Fig. 6.8, transforms were observed depending on the
time the DNA was extracted from the cells.
1. Time 1, the DNA is still outside the cells and accessible to the
DNase. No
Arg+ transformants are observed because the Arg+
donor DNA is all destryed by DNase.
2. Time 2, some of DNA is now inside the cells, where it can not
be degraded by the DNase, but this DNA is single-stranded.
It has not yet recombined with bacterial chromosomal DNA, and
so no Arg+ transformants observed in step 4.
3. Time 3, when some of the DNA has recombined with bacterial
chromosomal DNA, and so is again double-stranded, do
transformants appear in step 4.
■ Thus, the transformingf DNA enters the eclipse period for a
short time after it is added to competent cells, as expected if it
enters the cells in a single-stranded state.
Neither plasmids nor phage DNAs can be efficiently introduced
into naturally competent cells for two reasons:
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1. They must double stranded to replicate.
Natural transformation
requires breakage of double-stranded DNA and degradation of
one of the two strands so that a linear single strand can enter
the cells.
2. They must recyclize. However, pieces of plasmid or phage DNA can not
recyclize if there are no repeated or complementary sequences at their ends.
- To overcome the problem, they are usually dimerized and
multimerized into long concatemers.
- If a dimerized plasmid or phage DNA is cut only once, it still
has complementary sequences at its ends that can
recombine to recyclize the plasmid.
- Evidences to support: Most preparations of plasmid or phage DNAs contain
some dimers.
Role of natural transformation
1. Nutrition – Organisms may take up DNA for use as a carbon and nitrogen
source.
2. Repair – Cells may take up DNA from other cells to repair damage to their own
DNA.
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