Compiled & Prepared by,K.P.Senthil kumar.,M.Sc.,M.Phil.,ADAB

Tobacco plant

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Viral cells with luciferase

Fireflies glow in summer night – “ Luciferase

Activity ”

Aids molecular biologists interested in

mammalian gene transcription

Gut of firefly beetle and some marine organisms

it is present

Luciferase Activity not in any other eukaryotes

Excellent Reporter gene in promoter analysis

Derived from luc/lux gene of firefly - Photinus

pyralis

Enhance Research in Chemiluminescence and

Bioluminescence

Modified luciferase genes are constructed

Higher level of expression

Different assay reagents – Change the kinetics

of light release

Use of small molecules to stable the enzyme

Isolation and expression in different organisms

Luciferase gene – cDna

Peptide sequence at carboxy terminal

Targets the protein to peroxisome of the cell

Eliminated peroxisome targeting sequences

Elimination of sequences predicted to give

Rna secondary structure

Inclusion of optimal translation initiation

sequence

Swapping of prevalent insect codons for

mammalian counterparts

Inclusion of polyadenylation sequences

upstream and downstream from cDna of

Luciferase gene

Luciferase has characteristic behavior

Substrate specificity

Light release kinetics

Allosteric modulation

Intracellular stability

Presence of Mg2+ is essential

ATP + Luciferin + O2

AMP + Oxyluciferin + Ppi + light (560 nm)

pGL series available from Promega

Other luciferase genes isolated from diverse

marine and bacterial organisms

From sea pansy – Renilla reniformis

Different substrate and different biochemical

properties

Dual reporter assay systems

Optimum assay components differ from

luciferase from species to species

pGL series available from Promega

1. pGL3-Control vector

2. pGL3-Basic Vector

3. pGL3-Promoter Vector

4. pGL3-Enhancer Vector

Enhancer 2013-2249

Promoter 0048-0250

RVprimer3 is especially useful for identifying

positions of nested deletions.

Note: All three primers can be used for dsDNA

sequencing, but only

RVprimer4 and GLprimer2 also may be used for

ssDNA sequencing.

Acetyl CoA : CAT, widely used as Reporter

Indirect assay of transcriptional regulatory elements

Transfected Mammalian cells

Covalently modifies chloroamphenicol

Transfer an acetyl group from acetyl CoA to

primary hydroxyl residue C3-chloroamphenicol

Then to C1 and one more in C3 form 1,3-diacetyl

chloroamphenicol.

CAT – Responsible for resistance to

Chloroamphenicol; An antibiotic

Bind to peptidyl transferase center of

prokaryotic ribosomes

CAT – Available in plasmids of Gram +ve and

Gram – ve organisms.

Trimers consists of identical subunits ;

Mw 25 kDa

Trimer- β pleated sheets extends from 1

subunit to next.

Two substrates

Chloroamphenicol

Acetyl CoA

Approach active site through tunnels

Located in opposite sides of the molecule

Active site located at subunit interface

His residue – act as general base catalyst in

acetylation reaction

Expression of CAT in Mammalian cells:

Plasmid pSV2CAT

SV40 promoter/enhancer

29 bp of Un Translated Sequence

CAT coding sequence

8 bp of Dna 3’ to the UAA stop codon.

To assay putative promoters in mammalian cells

A derivative of pSV2CAT is constructed with

pSV0CAT

Promoter of SV40 replaced with the one under

test.

No endogenous DNA in eukaryotes

No competing activities

Enzyme is stable

Some assays measure in cell lysate

Expensive and Time consuming

Extracts prepared from C-14 labeled

Chloroamphenicol

Modified product separated from unmodified

drugs by TLC

Quantified by Autoradigraphy

Sensitivity increased if condition of extract adjusted

10 mM EDTA

Heated at 60˚C

10 minutes incubation

Mixture extracted with ethyl acetate

Partition in organic phase

Acetyl CoA remains in aqueous phase

liquid Scintilation counter

Express CAT activity in product formed per mg cell

extract per unit time.

Green Fluorescent Protein

Bioluminescent jellyfish Aequorea victoria

Emits characteristic Green Fluorescence

Activity of two proteins

Calcium-binding photoprotein “aequorin” and

its companion

Green Fluorescent Protein

GFP most important protein in BC and Mob.

Tool to understand and manipulate

Relation between protein structure and

intracellular process

From bacteria to mice

238 – residue peptide

Mw – 26,888 Da

Three Exons spread over 2.6 kb

Protein is stable even at

Heat

Extreme pH

Chemical denaturants

Continues to emit fluorescence after fixation in

formaldehyde.

Fluorescence is rapidly quenched under reducing

conditions.

The emission spectrum peaks at 508 nm

Distinct from chemiluminescence of aequorin i.e.,

blue – peaks near 470 nm.

Structure

β – barrel structure composed of 11 strands

, encapsulates 1α-helix.

Chromophore formed ; cyclization of residues

Ser-65

Tyr-66

Gly-67

contained in short helical structure

Buried inside β – barrel

Insulation gives greater resistant to denaturants

Function:

No separate biosynthetic pathway is required

Autocatalytic intramolecular reaction to create the

chromophore

Post translation occurs

Cyclization

Oxidation of the trimer Ser-65, dehydro Tyr-66,

Gly-67

Absorb light maximally at 390 nm

However intact GFP emits green light

Peak at 508 nm

Shoulder at 540 nm

It can be formed in wide range of cells that

normally do not produce light

Sensitive to

pH

Temperature

Ionic Strength

Chromophore may exists in two forms

Two peaks of absorbance

390 nm – protonated tyrosyl hydroxyl groups

480 nm – deprotonated tyrosyl hydroxyl

groups

GFP as Reporter:

Ability to fluoresce in organisms other than

aequorea

No other agents; such as

Abs

Cofactor

Enzyme – substrates required for its activity.

GFP used as reporter in

Caenorhabditis elegans

Bacteria and Yeasts

Drosophila

Zebra fish; Plants ; Cultured mammalian cells

Transgenic mice

Potential difficulties in heterologous expression :

Post translation modification requires > 1 hr

This delays the emission ability

Immediate readout is not possible

Efficient expression in higher organisms

Optimization of coding sequence required

No definite prediction of fusion protein

i.e., Function protein to analyze and GFP

A variety of constructs generated

Over expression of protein in some cells like

yeast

Confocal microscopy, Careful choice of

filters are required

GFP exhibits significant spectral change

Protein concentration

Ionic strength

pH

Important to assay under standard

condition

The entire cDNA sequence encoding GFP has

been

Mutagenized

Synthesized in several different ways

various groups to alter

Signal produced by reporter has been

increased considerably

Low level transcription is addressed

Insertion of strong constitutive promoters

Cytomegalovirus

SV40

HIV

Long terminal repeats upstream of the GFP-

coding region

Extensive structure driven ,site-directed

mutagenesis.

Variants each having altered fluorescence

excitation and/or emission spectra

Altered properties provide significant

advantage over wild type GFP

Mutation by substitution at Tyr-66 ;

generates

Fluoresce yellow

Blue

Cyan

Mutation at

Tyr-66 cause 20% reduced fluorescent

output

Thr-65 cause 4 to 6 folds greater than

wild type GFP.

E.coli β Galactosidase – 465,412 Da

Tetramer of 4 identical polypeptide subunits

1023 amino acids

Encoded by first gene of the Lac operon – lac Z

The individual polypeptide chain folds into 5

sequential domains

An extended section of 50 aminoacid residues at

the amino terminus

This is α peptide

β Galactosidase whose synthesis is induced by

lactose and other galactosides

Catalyzes two enzymatic reactions

Hydrolysis of β-D-galactopyrinosides

Lactose into glucose and galactose

A transgalactosidation reaction

Lactose is converted to allolactose, true

inducer of lac operon

β Galactosidase interact with series of analogs

of lactose

Glucose is replaced with other moieties

ONPG o-nitrophenyl-β-D-galactoside

X-gal

MUG 4-methyl umbelliferyl- β-D-galactoside

TPEG p-aminophenyl- β-D-thio-galactoside - INHIBITOR

PECULARITY:

The aminoacid and carboxyl domains of the

enzymes can be in different molecule

Two inactive fragments of the polypeptide chain

One lacking amino-terminal region (α acceptor)

Other lacking carboxy terminal region (α donor)

Vector

Both able to associate in vivo and in vitro

To form Tetrameric active enzyme

This unusual form of Complementation is α-

Complementation

Hydrolysis of ONPG

Spectrophotometric Assay

Cleaves β- galactosidase linkages

Hydrolysis synthetic ONPG into o-nitrophenol

Yellow in aqueous solution

Absorbance at 420 nm

bacterial cells +

permeabilized toluene or chloroform +

buffer with high concentration of β mercaptoethanol +

Incubation with ONPG ;

Reaction terminated with Sodium carbonate

OD 420 (o-nitrophenol + bacterial debris)

Remove the debris (centrifugation)

OD 420

Units of β galactosidase

= 1000 OD 420 /t v OD 600 Miller units

t- time of the reaction in minutes

v-volume of the culture in ml used for assay

OD 600 Absorbance at 600 nm of bacterial

cells just before enzyme assay

Induced culture contains almost 1000 units of β

galactosidase

Uninduced <1 unit

β galactosidase can also be expressed in mammalian

cells

Reporter gene technology:

To define a gene with readily measurable phenotype

Can be distinguished easily over a background of

endogenous proteins

Reporters selected :

Sensitivity

Dynamic range

Convenience

Reliability of their assay

Controlling the activity of genes by cis – reguation

sequences (Response Elements)

Responsive to alterations in Gene regulations and

Expression in host cells

Hormones and Growth Factors stimulate Target

cells

1.The cAMP response element (CRE) interacts

with CREB (CRE-binding protein), which is

regulated by cAMP

2.Estrogen response element (ERE) are the

recognition sites of estrogen receptor

3.Glucocorticoid response element (GRE) and

glucocorticoid receptor

Note that hormones are not transcription factors, but

many of their receptors

4. Heat shock response element (HSE) is present in

heat shock protein genes.

In response to external stress (e.g. high temperature),

the heat shock factor (HSF) will interact with HSE,

stimulating expression of heat shock proteins.

5. Serum response element (SRE) binds to serum

response factor (SRF), which can be activated by

many growth factors in serum.

The Fos subunit of AP-1 is encoded by a gene

containing SRE. Fos is known to play an important

role in cell cycle progression.

Reference:

Sambrook J. et al. Molecular Cloning: A Laboratory

Manual. New York; Cold Spring Harbor 1989.

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