MICR 201 Microbiology for Health Related Sciences - Cal State LA ...

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Feb 20, 2013 (4 years and 8 months ago)

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Lecture 4:Microbial genetics, biotechnology, and recombinant DNA

Edith Porter, M.D.

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Microbial genetics


Genotype and phenotype


DNA and chromosomes


Flow of genetic information


DNA replication, RNA and Protein synthesis


Bacterial gene regulation


Mutations


Gene transfer and recombination


Biotechnology and recombinant DNA


Recombinant DNA technology


Techniques in gene modification


Applications or recombinant DNA




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Science of heredity


Study of genes, how genes
carry information, how
genes can be transferred,
how the expression of the
encoded information is
regulated, how genes render
specific characteristics to the
organism that harbors these
genes


Genotype: collection of
genes


Phenotype: collection of
proteins encoded by these
genes


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A gene is a specific sequence of nucleotides along
the DNA
strand


Consists of a
promotor
, coding and terminator
region







A
gene can code for


mRNA (used to make proteins from amino acids at
ribosomes
)


rRNA

(synthesized in the nucleolus in
eukaryotes
)


tRNA

(brings specific single amino acids to the
ribosomes
)


Binds RNA
-
polymerase Indicates end of gene

Promoter

Terminator

Coding region

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Sequence of nucleotides


Base: Adenine, thymine,
cytosine, and guanine


Deoxyribose


Phosphate


Double helix associated with
proteins


Strands held together by
hydrogen bonds between
AT and CG


Strands antiparallel

6


E. coli

DNA ~ 1300
m
m, the average cell ~
2
-
4
m
m


Eukaryotic DNA ~ 1.8 m (= 1,800,000
m
m),
the average cell ~ 15
-
30
m
m


Supercoiling


Requires special enzymes to


Supercoil


Relax supercoiling (topoisomerases; e.g. gyrase
in prokaryotes)


Unwind (helicases)


Proteins to stabilize


Histones in eukaryotes


Histone
-
like proteins in prokaryotes

Ciprofloxacin:

Gyrase

inhibitor

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8

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Transfer of the genetic information to the next generation


1 strand remains the parent strand, 1 strand is newly
synthesized


Mistakes only in 1/ 10
10

bases!


Direction


In eukaryotes: uni
-
directional


In prokaryotes: circular genome and bi
-
directional replication

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Origin may be
attached to the
cell membrane

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To copy DNA into RNA (synthesis of complimentary strand of
RNA from a DNA template)


RNA consists of base ribose and phosphate, single stranded


Messenger RNA (mRNA)


Information for proteins


Thymine replaced with uracil


Transfer RNA (tRNA): carries single specific amino acid residues


Thymine in tRNA in eukaryotes and bacteria


No thymine in archaea in tRNA


Ribosomal RNA (rRNA): assists mRNA in binding to the ribosome


Transcription begins when RNA polymerase binds to the
promotor sequence


Transcription proceeds in the 5
'



3
'

direction


Transcription stops when it reaches the

terminator sequence



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Protein synthesis


Nucleotide
language encoded
within mRNA is translated into
amino acid language


mRNA is translated in
codons


One
codon

consists of three
nucleotides


One
codon

codes for one amino acid


Translation of mRNA begins at the
start
codon
: AUG


Translation ends at a stop
codon
:
UAA
,
UAG
,
UGA


tRNA

has
anticodons

complementary to the mRNA
codons

The universal (degenerative) genetic code

13

In bacteria, first amino acid is always formyl methionine

14

Elongation is
target for many
bacterial toxins
and antibiotics!

15


Usually a number of
ribosomes are attached
to one mRNA molecule


Multiple protein copies
from one mRNA
molecule


16


Different enzymes


In eukaryotes exons, introns, repetitive sequences


Introns are transcribed but not translated nucleotide sequences


Cut out by ribozymes (RNA with enzymatic activity)


In prokaryotes exons only


Exceptions: archaea and cyanobacteria


In eukaryotes mRNA must exit nucleus and therefore must
be completed before translation can begin


In prokaryotes simultaneous transcription and translation


Gene overlap


Never in eukaryotes, sometimes in prokaryotes, often in viruses


Gene 1

Gene 2

Gene 3

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Of all genes 60


80% are
constitutive

(always
expressed)


20


40% are
regulated

(expressed only when
needed)


One form of gene
regulation
is negative regulation
by means of operators
and
repressors inserted
between the promoter and coding gene region






RNA
-
polymerase cannot bind to promoter or
cannot proceed when operator is occupied by
repressor


The unit consisting of a promoter, operator and the
structural gene is called
operon

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Binds RNA
-
polymerase Indicates end of gene

Promoter

Terminator

Coding region


An operon consists of promoter, operator and
the associated structural genes that need to
be regulated

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During base line metabolism


Operator is occupied by an
active repressor


Gene is turned off


When needed


Inducer

binds to active repressor


Repressor is inactivated


Repressor cannot bind anymore to operator


RNA

polymerase can bind to promoter and proceed with
transcription


Gene is turned on


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During base line metabolism constant need of
gene product


Operator is not occupied by a repressor


Inactive repressor
cannot bind to operator


RNA

polymerase binds to promoter and proceed with
transcription


Gene is turned on


When gene product is not needed anymore


Co
-
repressor

(typically the gene product) binds to the
inactive repressor


Repressor is activated


Now repressor can bind to operator


Gene is turned off

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Mutations


Gene transfer and recombination

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Not
-
corrected errors during DNA replication


Occur spontaneously rarely at 1/10
9
replicated base pairs


Lead to permanent changes in genotype


If coupled to changes in proteins with altered function: changes in
phenotype


Base substitutions (point mutations) can lead to


Missense
: one amino acid change with major consequences


A

T leads to glutamic acid


valine in hemoglobin: sickle cell disease


Nonsense
: can lead to stop of transcription


Deletion or insertion of a few base pairs


Frame shift mutation
: shift translational reading frame, major
alterations in amino acid sequence, almost always dysfunction protein
results


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Increased antibiotic resistance or loss of
antibiotic resistance


Increased pathogenicity or loss of
pathogenicity


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Natural mutation rate is ~ 1 in
10
9

replicated base pairs (or in 10
6

replicated genes)


Mutagens increase
the rate of
mutations by factor 10


1000


Chemical


Point mutations


Nitrous acid


Nucleosid

analogs


Frame shift
mutations


Benzpyrene

(smoke)


Aflatoxin

(
Aspergillus

flavus

toxin)


Physical


UV
-

radiation


Thymine
dimerization


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Auxotrophic mutants


Cannot grow without the presence of a
particular nutrient, e.g. histidine


When exposed to mutagens development of
revertants


Can grow in the absence of this nutrient


Assay performed with addition of liver extract


Some mutagens are only formed after
metabolisation by liver

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Vertical transfer


Passing genes to off springs


Horizontal transfer


Passing genes laterally to representatives of
the same generation


Donor cell passes genes which will be
integrated into recipient’s DNA

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Transformation


Uptake of naked DNA


Conjugation


Plasmid uptake through Sex
-
Pili


Requires cell to cell contact and two mating
types


Transduction


Uptake of foreign DNA through a
bacteriophage

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DNA
replication
DNA



DNA


In bacteria, bi
-
directional


Transcription: DNA


RNA


Translation: RNA


Protein


In bacteria, transcription and translation occur simultaneously


Bacterial gene
regulation
utilizes
operons


Inducible genes


Repressible genes


Mutations are permanent, inheritable changes of the genetic
informati0n


Missense

(protein with altered amino acid sequence may result)


Nonsense (protein synthesis is aborted)


Frameshift

(entirely different protein results)


Mutagens increase the
frquency

of mutations


Genetic transfer and recombination can be achieved by


Transformation (uptake of naked DNA)


Conjugation (uptake via cell to cell contact and sex
pili
)


Transduction (genetic exchange via a
bacteriophage
)



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Biotechnology: the use of microorganisms,
cells, or cell components to make a
product that is not naturally produced


Foods, antibiotics, vitamins, enzymes


Recombinant DNA technology: insertion
or modification of genes to produce
desired proteins

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Genetic engineering


Technique for artificial DNA recombination


Examples:


Higher vertebrate genes (animal including human)
inserted into a bacterial genome


Human growth hormone gene inserted into
E. coli


Viral gene into yeast cells


Hepatitis B gene inserted into yeast cells for vaccine production

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DNA with the gene of interest


Selection


Mutation


Vector DNA


Restriction enzymes


Discovered when studying viruses


Some bacteria can degrade viruses with these enzyme and are
protected against these viruses


Cut at certain nucleotide sequences


Recognize 4, 6, or 8 base pairs


Produce “sticky ends”


Ligases to join the DNA fragments

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Self replicating DNA


Must not be destroyed by recipient cell


Circular DNA like
plasmids


Virus

which is rapidly integrated into host genome


Vectors contain marker genes


Tag to identify vector


Often antibiotic resistance genes or enzyme carrying out easily
identifiable reactions


Can be used for cloning


Shuttle vectors


Can exist in several different species


Bacteria, yeasts, mammals


Bacteria, fungi, plants

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To make numerous (unlimited) identical
copies of one original


Cell cloning: 1 single cell multiplied


Gene cloning: 1 single gene is inserted into
a vector and replicated as the vector is
replicated

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Marker

Genes

Ampicillin

Resistance

Beta
-
galactosidase

Restriction

Enzyme Sites

Origin of Replication
for Independent
Replication

Vector Name

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Beta
-
galactosidase

inactivated

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Agar with Ampicillin and
X
-
gal (substrate for
beta
-
galactosidase)


DNA can be inserted
into a cell by:


Transformation (naked
DNA in solution)


Transduction (via virus)


Electroporation


Gene gun


DNA coated gold bullets


Microinjection

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DNA fingerprinting


PCR reaction

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Identical DNA will generate
identical DNA fragments
when subjected to
restriction enzyme
digestion


Subject DNA to agarose gel
electrophoresis and
compare DNA fragment
pattern (restriction
fragment length pattern)

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To quickly specifically amplify small samples
of DNA


From 1 copy to 1 billion copies within hours


25 to 35 reaction cycles


High specificity


High sensitivity


Not a functional assay

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Original DNA (purified or cDNA made from
RNA via reverse transcription)


DNA polymerase


taq

polymerase


From thermophile bacterium
Thermus aquaticus


Heat stable, functions at ~ 72

C


Primers (complementary short nucleotide
sequences matching the beginning/end of DNA
of interest)


Nucleotides


Appropriate buffer


Thermocycler

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1.
Denaturing by heat


Separate DNA strands
at ~ 95

C



2.
Annealing


Primers attach at
~50


60

C



3.
Extension


Polymerase extends
DNA strand at ~72

C

55


In clinical diagnostics


Organism is hard or not to culture


Very low numbers of organism are present


In research

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Subunit vaccines against infectious diseases


HPV (virus coat)


Gene therapy


Introducing functional genes into defective
genome


Gene silencing via inhibitory RNA (short
interfering RNA, double stranded)


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PCR results of patient samples

1: bp size ladder; 2:negative control;

3
-
8: patient samples

Virus specific


Recombinant DNA technology


Artificial

DNA recombination between
unrelated species


Insertion of new genes into cells


Typically requires restriction enzymes and
vectors


Cloning: to amplify a gene in another cell


PCR (polymerase chain reaction)


To specifically detect and amplify small
samples of
DNA

60


The method of using RFLPs to identify


bacterial or viral pathogens is called



a. Proteomics


b. DNA fingerprinting


c. Genetic screening


d. DNA sequencing


The use of an antibiotic resistance gene on a
plasmid used in genetic engineering makes



Direct selection possible.


The recombinant cell dangerous.


Replica plating possible


The recombinant cell unable to survive