:Contents
introduction-
.transfer of DNA within bacterial cells-
.transfer of DNA between bacterial cells-
.conjugation.1
.transduction.2
.Transformation.3
.recombination-
:Introduction
.The genetic material of a typical bacterium
Escherichia Coli , consists of a single circular DNA molecule
with a molecular weight of about 2x10^9 and is composed
of approximately 5x10^6 base pairs. This amount of genetic
information can code for about 2000 proteins with an
.average molecular weight of 50,000
The DNA of the smallest free-living organism , the wall-less
bacterium Mycoplasma , has a molecular weight of 5x10^8 .
The DNA of human cells contains about 3x10^9 base pairs
.and encodes about 100,000 proteins
Note that bacteria are haploid while eukaryotic
cells ( such as human cells ) are diploid . In diploid
cells, one copy of a gene ( allele ) may be expressed
as a protein ( i.e. , be dominant ) , whereas another
.allele may not be expressed ( i.e. , be recessive )
In haploid cells , any gene that has mutated – and
therefore is not expressed – results in a cell that
.has lost that trait
:Mutations
A mutation is a change in the base sequence of
DNA that usually results in insertion of a different
amino acid into a protein and the appearance of an
.altered phenotype
: Mutations result from three types of molecular changes
Base substitution.1
This occurs when one base is inserted in place of another. It
takes place at the time of DNA replication, either because
the DNA polymerase makes an error or because a mutagen
alters the hydrogen bonding of the base being used as a
.template in such a manner that the wrong base is inserted
When the base substitution results in a codon that simply
causes a different amino acid to be inserted , the mutation is
called missense mutation , when the base substitution
generates a termination codon that stops protein synthesis
prematurely , the mutation is called a nonsense mutation.
.Nonsense mutations almost always destroy protein function
Frameshift Mutation.2
This occurs when one or more base pairs are added
od deleted , which shifts the reading frame on the
ribosome and results in incorporation of the wrong
amino acids “downstream” from the mutation and
.in the production of an inactive protein
: Transposons
The third type of mutation occurs when
transposons or insertion sequences are integrated
. into the DNA
These newly inserted pieces of DNA can cause
profound changes in the genes into which they
. inserted and in adjacent genes
Mutations can be caused by chemicals , radiation ,
. or viruses
Chemicals (A)
: act in several different ways
Some , such as nitrous acid and alkylating agents(1)
, alter the existing base so that it forms a hydrogen
bond preferentially with the wrong base (eg.,
adenine would no longer pair with thymine but
.with cytosine )
Some chemicals , such as 5-bromouracil , are base(2)
.analogues , since they resemble normal bases
Because the bromine atom has an atomic radius similar
to that of methyl group , 5-bromouracil can be inserted
.in place of thymine ( 5-methyluracil )
However , 5-bromouracil has less hydrogen-bonding
fidelity than does thymine , and so it binds to guanine
with greater frequency . This results in a transition from
an A-T base pair to a G-C base pair, thereby producing a
mutation. The antiviral drug iododeoxyuridine acts as a
.base analogue of thymidine
Some chemicals , such as benzpyrene , which is(3)
found in tobacco smoke , bind to the existing DNA
bases and cause frameshift mutations . These
chemicals which are frequently carcinogens as well
as mutagen , intercalate between the adjacent
bases , thereby distorting and offsetting the DNA
. sequence
: X- rays and U.V light can cause mutations also (B)
X-rays have high energy and can damage DNA in three
: ways
By breaking the covalent bonds that hold the ribose(1)
.phosphate chain together
.By producing free radicals that can attack the bases(2)
By altering the electrons in the bases and thus(3)
.changing their hydrogen bonding
U.V radiation , which has lower energy than x- rays
, causes the cross- linking of the adjacent
pyrimidine bases to form dimers. This cross- linking
(dimer eg., of adjacent thymines to form a thymine)
.results in inability of the DNA to replicate properly
: Viruses (c)
Certain viruses , such as the bacterial virus Mu
(mutator bacteriophage) , cause a high frequency
of mutations when their DNA is inserted into the
bacterial chromosome . Since the viral DNA can
insert into many different sites , mutations in
either various genes can occur. These mutations are
. frameshift mutations or deletions
: Conditional lethal mutations
Conditional lethal mutations are of medical interest because
.they may be useful in vaccines (eg., influenza vaccine )
The word conditional indicates that the mutation is
.expressed only under certain conditions
The most important conditional lethal mutations are the
temperature sensitive ones. Temperature – sensitive
organisms can replicate at a relatively low , permissive
temperature (eg., 32c ) but cannot grow at a higher ,
restrictive temperature ( eg., 37c ). This behavior is due to
function a mutation that causes an amino acid change in an
essential protein , allowing it to function normally at 32c but
not at 37c because of an altered conformation at the higher
. temperature
An example of a conditional lethal mutant of
medical importance is a strain of influenza virus
currently used in an experimental vaccine . This
vaccine contains a virus that cannot grow at a 37c
and hence cannot infect the lungs and cause
pneumonia , but it can grow at 32c in the nose ,
. where it can replicate and induce immunity
: Transfer of DNA within Bacterial cells
: Transposons
Transposons transfer DNA from one site on the
bacterial chromosome to another site or to a plasmid.
They do so by synthesizing a copy of their DNA and
inserting the copy at another site in the bacterial
. chromosome or the plasmid
The transfer of a transposon to a plasmid and the
subsequent transfer of the plasmid to another
bacterium by conjugation contributes significantly to
,the spread of antibiotic resistance
Transfer of DNA within bacteria also occurs by programmed
rearrangements . These gene rearrangements account for
many of the antigenic changes seen in Neisseria
gonorrhoeae and Borrelia recurrentis , the cause of
.relapsing fever
A programmed rearrangement consists of the movement of
a gene from a silent storage site where the gene is not
expressed to an active site where transcription and
translation occur . There are many silent genes that encode
variants of the antigens , and the insertion of a new gene
into the active site in a sequential , repeated programmed
.manner is the source of the consistent antigenic variation
These movements are not induced by an immune response
.but have the effect of allowing the organism to evade it
: Transfer of DNA between bacterial cells
: By three methods
.conjugation.1
.transduction.2
.transformation.3
From a medical viewpoint , the most important
consequence of DNA transfer is that antibiotic
resistance genes are spread from one bacterium to
.another by these processes
: Conjugation.1
Conjugation is the mating of two bacterial cells , during
cell which DNA is transferred from the donor to the recipient
.
The mating process is controlled by an F ( fertility ) plasmid (
F factor ) , which carries the genes for the proteins required
.for conjugation
One of the most important proteins is pilin , which forms the
. sex pilus ( conjugation tube )
Mating begins when the pilus of the donor male bacterium
carrying the F factor (F+ ) attaches to a receptor on the
surface of the recipient female bacterium , which does not
.contain an F factor (F -)
The cells are then drawn into direct contact by
“reeling in “ the pilus . After an enzymatic cleavage
of the F factor DNA , one strand is transferred
across the conjugal bridge into the recipient cell .
The process is completed by synthesis of the
complementary strand to form a double-stranded F
cells. factor plasmid in both the donor and recipient
The recipient is now an (F +) male cell that is
. capable of transmitting the plasmid further
Note that in this instance only the F factor , and not
. the bacterial chromosome , has been transferred
into Some (F +) cells have their F plasmid integrated
the bacterial DNA and thereby acquire the
capability of transferring the chromosome into
another cell. These cells are called Hfr ( high –
. frequency recombination ) cells
During this transfer , the single strand of DNA that
enters the recipient (F -) cell contains a piece of the
bacterial F factor at the leading end followed by the
chromosome and then the remainder of the F
factor . The time required for complete transfer of
. the bacterial DNA is approximately 100 mins
portion Most matings result in the transfer of only a
of the donor chromosome because the attachment
between the two cells can break . The donor cell
genes that are transferred vary since the F plasmid
can integrate at several different sites in the
bacterial DNA . The bacterial genes adjacent to the
leading piece of the F factor are the first and
therefore the most frequently transferred. The
newly acquired DNA can recombine into the
recipients DNA and become a stable component of
.its genetic material
: Transduction.2
of Transduction is the transfer of cell DNA by means
.a bacterial virus ( bacteriophage , phage )
During the growth of the virus within the cell , a
piece of bacterial DNA is incorporate into the virus
particle and is carried into the recipient cell at the
. time of infection
Within the recipient cell , the phage DNA can
integrate into the cell DNA and the cell can acquire
.a new trait- a process called lysogenic
This process can change a nonpathogenic organism
into a pathogenic one . Diphtheria toxin , botulinum
toxin , cholera toxin , and erythrogenic toxin (
streptococcus pyogenes ) are encoded by
bacteriophages and can be transferred by
.transduction
There are two types of transduction , generalized
: and specialized
: The generalized type
Occurs when the virus carries a segment from any
part of the bacterial chromosome . This occurs
because the cell DNA is fragmented after phage
infection and pieces of cell DNA the same size as
the viral DNA are incorporated into the virus
particle at a frequency of about 1 in every 1000
.virus particles
: The specialized type
Occurs when the bacterial virus DNA that has
integrated into the cell DNA is excised and carries
. with it an adjacent part of the cell DNA
Since most lysogenic (temperate) phages integrate
at specific sites in the bacterial DNA , the adjacent
cellular genes that are transduced are usually
.specific to that virus
: Transformation.3
Transformation is the transfer of DNA itself from one
cell to another . This occurs by either of the two
: following methods
In nature , dying bacteria may release their DNA ,(1)
which may be taken up by recipient cells. There is little
evidence that this natural process plays a significant
. role in disease
In the laboratory , an investigator may extract DNA(2)
from one type of bacteria and introduce it into
.genetically different bacteria
When purified DNA is injected into the nucleus of a
. eukaryotic cell , the process is called transfection
Transfection is frequently used In genetic engineering
. procedures
The experimental use of transformation has revealed
important information about DNA . In 1994 , it was
shown that DNA extracted from encapsulated smooth
pneumococci could transform nonencapsulated rough
.pneumococci into encapsulated smooth organisms
This demonstration that the transforming principle was
DNA marked the first evidence that DNA was the
.genetic material
: Recombination
Once the DNA is transferred from the donor to the
recipient cell by one of the three processes just
described , it can integrate into the host cell
. chromosome by recombination
: There are two types of recombination
Homologous recombination , in which two (1)
pieces of DNA that have extensive homologous
regions pair up and exchange pieces by the
.processes of breakage and reunion
Nonhomologous recombination , in which little (2)
. of any , homology is necessary
Different genetic loci govern these two types , and
so it is presumed . Although is known that a variety
of endonucleases and ligases are involved , the
. precise sequence of events is unknown
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