BACTERIAL GENETICS

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11 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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BACTERIAL GEN
ETICS

Learning objectives:

At the end of the lecture the student should be able to:



Enlist the methods of DNA transfer in microorganisms.



State the significance of DNA transfer in drug resistance.



Enlist & describe the types of mutations in ba
cteria.



Describe the process of lysogeny.



Describe the role of mutations in drug resistance in infectious diseases


Understanding Genetics



We resemble and differ because of Genetic configurations



Parents
-

Son
-

Daughter, how they resemble each other.

They

breed true from Generation to Generation

But vary in small proportions in progeny.

Bacteria too obey the laws of Genetics








DNA

A Complex Structure Makes Life











Knowledge on DNA lead to advances in Molecular Biology



Central dogma of Life


D
eoxyribonucleic acid



DNA carries the g
enetic information



DNA is transcribed to RNA


Polypeptides


Cell Function depends upon specific polypeptides


Proteins


Enzymes


DNA is a store house of Protein synthesis


DNA acts a Template for synthesis

of mRNA


Virus differs from other as they contains either DNA or RNA


The Bacterial Genome



Chromosomes



Contains a Double stranded molecules of
DNA arranged in circular form.



Length 1
,000

microns.



Bacterial DNA contains about 4,000

kilobases



Bacte
ria are haploid: they have a single
chromosome and therefore a single copy of
each gene.



In haploid cells, any gene that has mutated


and is therefore not expressed


results in
a cell that has lost that trait.



Plasmids



Plasmids are circular DNA molecul
es
present in the cytoplasm of the Bacteria



Capable of Autonomous replication



Can transfer genes from one cell to other



Act as vectors in Genetic engineering.



Can also present in Yeasts





Plasmids




Plasmid seem to be ubiquitous in bacteria, May encode
g
enetic information for properties




Resistance to Antibiotics




Bacteriocins production




Enterotoxin productions




Enhanced pathogen
city




Reduced Sensitivity to

mutagens




Degrade complex organic molecules






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:



1) Base substitution



2) Frameshift mutation



3) Transposons or ins
ertion sequences



Mutations can be caused by chemical, radiations or viruses



Mutation is a Random, Undirected, Heritable variation


Transfer of DNA within Bacterial cells



Transposons



Programmed rearrangement



These gene rearrangements account for many of the

antigenic changes
seen in Neisseria gonorrhoea and Borrelia recurrentis the cause of
relapsing fever.



A programmed rearrangement consist of movement of a gene from a silent
storage site where the gene is not expressed to an active site where
transcription

and translation occur. 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 have the effect of allowing the organism to evade the
immune response.




TRANSPOSONS



Mobile genetic elements



The arrangement of g
enes in the
genome of bacteria
and probably all
organisms is not entirely static. Certain
DNA segments called transposons have
the ability to move place to place on the
chromosome and into and out

of
plasmids.



Mobile genetic elements are probably
responsible for most of the genetic
variability in natural bacterial
populations, and for the
spread of
antibiotic resistance genes
.









Tra
nsformation of Genetic material
(Gene Transfer)



Occur by thr
ee methods:



1)
Conjugation



2) Transduction



3) Transformation



From a medical point of view the most important
consequence of a DNA transfer is that antibiotic
resistance genes are spread from one bacterium to
another by these processes





CONJUGATION



Conjug
ation is the process by which the bacteria
transfer genes from one cell to another by cell
-
to
-
cell
contact.



The process requires the presence on the donor cells
F+ of hair like projections called sex pili that make
contact with specific receptor sites on t
he surface of
recipient cells


High frequency recombinant Conjugation




When it exists as a free plasmid, the F plasmid can
only transfer itself. This isn’t all that useful for
genetics.



However, sometimes the F plasmid can become
incorporated into the
bacterial chromosome, by a
crossover between the F plasmid and the chromosome.
The resulting bacterial cell is called an “Hfr”, which
stands for “High frequency of recombination”.



Hfr bacteria conjugate just like F+ do, but they drag a
copy of the entire c
hromosome into the F
-

cell.










TRANSDUCTION



Transduction is defined as transfer of portion of
DNA from one bacterium to another by
Bacteriophages.



During the growth of virus within the cell a piece of
bacterial DNA is incorporated into the virus par
ticle
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
conversion



This process can change a non pathog
enic organism into a pathogenic one.



Bacteriophages



Are viruses that paras
itize bacteria and consists of n
ucleic acid core and a
protein coat



A phage particle may have at its core besides its own
nucleic acid and a segment of the Host DNA




















Specialized transduction



Only certain bacterial genes located in the bacterial chromosome in close
proximity to the prophage insertion site of the transducing phage are
transduced.






Generalised transduction



A random fragment of bacterial DNA resulti
ng from
phage induced cleavage of bacterial chromosome,
is accidentally in a phage protein

coat in place of
the phage DNA.

When this rare phage particle
infects a cell it injects the bacterial DNA fragment
into the cell, it becomes integrated into the
reci
pient chromosome by recombination.






Transformation



Is the transfer of DNA itself from one cell to
another.



In nature, dying bacteria may release their DNA
which may be taken up by recipient cells.



In the laboratory an investigator may extract DNA
from o
ne type of bacteria and inject 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






Griffith Ph
enomenon




Recombination

Once the
DNA is transferred from the dono
r to the recipient cell it can integrate
into the host cell chromosome by recombination.

1) Homologous recombination: in which two pieces of DNA
that has

extensive
homologous regions pair
up and exchange pieces by the process of breakage
and reunion

2) Non

homologous recombination in which little if any homology is necessary


What is Gene Therapy
?



Gene therapy

is the insertion of genes into an individual's cells and
tissues to treat a dise
ase, such as a hereditary disease in which a
deleterious mutant allele is replaced with a functional one. Although the
technology is still in its infancy, it has been used with some success.



Vivo to Vitro












What Gen
e therapy can
achieve
?



Replacing a mutated gene that causes disease
with a healthy copy of the gene.



Inactivating, or “knocking out,” a mutated gene that
is functioning improperly.



Introducing a new gene into the body to help fight
a disease.


Gene
tically
Engineered Products



Can prepare desired protein in pure form in
economic way


Somatostatin



Commercial preparations o
f


Cloned Human Insulin



Interferons


Hepatitis B vaccine







Restriction Endonucleases



A
restriction enzyme

(or
restriction
endon
uclease
) is an
enzyme

that
cuts double
-
stranded
DNA
. The enzyme makes two incisions, one through
each of the sugar
-
phosphate backbones (i.e., each strand) of the double
helix without damaging the nitrogenous
bases
. They work with cutting

up
foreign DNA



B
lotting Techniques



Drug fragments obtained by restriction enzyme digestion on separation Gel
can be transferred to Nitrocellulose or nylon membranes



Several methods


1 Southern blotting


2 Northern Blotting


3 Western blotting










Western Blot to confirm

HIV Infections made land mark Diagnostic
tool



Western Blot testing is confirmatory test for
diagnosis of HIV/AIDS



Identifies antibodies directed against
different antigens in pathogen


Surface,


Core


RT antigen










PCR
-
Sequences



PCR consists of several cycles of sequential
DNA replication where the products of first
cycle becomes the template for the Next



It makes available abundant quantities of
specific DNA sequences starting







Genetic Mapping



Genetic
sequences for Bacteriophages
and virus



Genetic mapping is done most of the
Human Genes


References

Lippincott’s Illustrated Reviews, Biochemistry

5th edition

Ch. 33 Biotechnology & Human Disease

Pgs. # 465
-
487