Chapter 8Chapter 8

Manipulating Proteins, Manipulating Proteins, DNA, and RNADNA, and RNA

WHY STUDY CELLS IN CULTURE?

• More homogeneous population

• Controlled experimental conditions

• Clonal isolates** - a genetically homogeneous population of cells arising from a single cell

•Assumption that response reflects what occurs at the unicellular level

WHY STUDY MICROORGANISMS LIKE BACTERIA, YEAST, AND VIRUSES?

• Easy and fast - will grow well on minimal medium (carbon source: glucose; nitrogen source: ammonium chloride; salts)

• When grown on a semisolid surface (eg. agar) can easily generate clonal isolates

•viruses have small genomes

CLONAL GROWTH

Bacterial colonies on agar plate

(each colony contains ~107 cells

after ~12 h)

Mixed bacterial culture

Clonal bacterial culture

• all bacteria are genetically identical

PREPARING A PRIMARY CULTURE OF ANIMAL CELLS• isolate a fragment of tissue of choice (eg. skin, muscle

• dissect away undesirable tissues and membranes• mince and digest the extracellular matrix (ECM) with one or more proteinases (eg. trypsin, collagenase)• isolate free cells (eg. by filtration or centrifugation) and plate onto petri dishes under appropriate growth medium•very rich media- 9 essential amino acids can not be synthesized by adult vertebrates: H,I,L,K,M,F,A,T,W,V. Medium must also contain C,Q and Y because these aa are made by specialized cells in the body and vitamins-SERUM-non cellular part of blood

ADVANTAGES and DISADVANTAGES of growing animal cells in culture

ADVANTAGES

• allows specific cell types to be studied free of the influence of surrounding tissues in the intact animal

• provides more control over experimental conditions

• can mimic cell-cell and cell-ECM interactions seen in tissues

• clonal colonies can be generated in ~ 2 weeks

• defined, serum-free medium formulations are available for some cell types

DISADVANTAGES

• question of cell behaviour in culture vs. in tissues

• can be difficult to grow or to maintain consistent growth conditions from one experiment to another

• growth medium is more complex - requires essential amino acids, vitamins, serum (hormones, growth factors, etc.)

ANIMAL CELLS IN CULTURE

Tissue culture flask

Growth medium

Gelatin or collagen substratum

Cells

37oC5% CO2

PRIMARY CULTURES

• best representation of cell behaviour in normal tissues

• have a finite lifespan (Hayflick limit - undergo replicative senescence after 50-60 generations (doublings; divisions)

• cell types commonly prepared include fibroblasts (skin), myoblasts (skeletal muscle), cardiomyocytes (heart)

TRANSFORMED CELLS

• can grow indefinitely in culture (have acquired one or more genetic mutations that allow them to escape senescence

• often these cells are less phenotypically related to the source tissue

• some can retain the ability to differentiate (eg. rodent muscle cell lines)

•examples include tumour cell lines (eg. HeLa cervical cancer cells established in 1952)

Two classes of animal cell cultures

NORMAL AND TRANSFORMED CELLS

EARLY MITOTIC SENESCENCE TRANSFORMATION

CARCINOMA

HYBRID CELL LINES (HETEROKARYONS)

• prepared by fusion of primary cells (human or mouse) with a transformed rodent (eg. hamster or mouse) cell line

•accomplished by co-incubating the two cell types with agents that promote cell membrane fusion (eg. polyethylene glycol (PEG), enveloped viruses) followed by some form of metabolic selection provided by the primary cells (eg. HAT medium: hypoxanthine (purine substrate for salvage pathway to produce guanylate); aminopterin (an antifolate that blocks the de novo purine synthetic pathway); thymidine (to provide for thymidylate synthesis))

• in human-rodent fusions, tendency is for the cells to lose human chromosomes - growth in selective medium that requires maintenance of a particular human chromosome can lead to the production of somatic cell hybrid panels containing defined human chromosomes for genetic mapping

•hybridoma = immortal cell line that produces a monoclonal (monospecific) antibody - produced from fusion of B-lymphocytes isolated from mouse spleens or lymph nodes (which together produce polyclonal antibodies following challenge with an antigen of interest), followed by clonal expansion and analysis of individual colonies for the production of the monoclonal antibody of interest

Common Cell TypesCommon Cell Types

Production of Hybrid CellsProduction of Hybrid Cells

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Generation of Monoclonal AntibodiesGeneration of Monoclonal Antibodies

Fractionation of CellsFractionation of Cells

Velocity and Equilibrium SedimentationVelocity and Equilibrium Sedimentation

ChromatographyChromatography

Matrices Used for ChromatographyMatrices Used for Chromatography

Elution Profiles Elution Profiles from different from different

matricesmatrices

SDS-PAGESDS-PAGE

SDS-PAGESDS-PAGE

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Isoelectric FocusingIsoelectric Focusing

Peptide Mapping of ProteinsPeptide Mapping of Proteins

intronexonpromoterenhancer

AAAAAAA

Untranslated region (UTR)

5’ 3’

5’3’

chromosome

gene

mRNA

FUNCTION

DNA

RNA

PROTEIN

Coding region

Untranslated region (UTR)

protein

CENTRAL DOGMA and GENE CLONING

FUNCTION

DNA

cDNA

PROTEIN

AAAAAAA

5’ 3’

5’3’

chromosome

gene

mRNA

protein

GENE CLONING: DNA to PROTEIN

MUTATION

DNA CLONING

A method for identifying and purifying a

particular DNA fragment (clone) of interest

from a complex mixture of DNA fragments, and

then producing large numbers of the fragment

(clone) of interest.

DNA CLONING TOOLS

RESTRICTION ENZYMES

VECTORS

DNA LIGASE

COMPETENT BACTERIAL CELLS

ANTIBIOTICS

DNA CLONING: RESTRICTION ENZYMES

RESTRICTION ENZYMES: Bacterial proteins (enzymes) that cut DNA molecules at specific sequences (endonucleases).

• restriction site: a specific 4- to 8-bp DNA sequences identified by a restriction enzyme

• restriction sites are typically short inverted repeat sequences

•restriction fragment: a piece of DNA that is released from a larger piece of DNA (eg. genomic DNA) following digestion with one or more restriction enzymes

• several hundred different restriction enzymes are known, each with its own unique restriction site

AAGCTT

TTCGAAHindIII

5’ 3’

5’3’

DNA CLONING: RESTRICTION ENZYMES

A AGCTT

TTCGA A

3’

5’

5’

3’

CCCGGG

GGGCCC

GGTACC

CCATGG

SmaI

KpnI

5’

3’

AAGCTT

TTCGAAHindIII

5’ 3’

5’3’

DNA CLONING: RESTRICTION ENZYMES

OVERHANGS

3’

5’

3’

5’

5’

3’

DNA CLONING: RESTRICTION MAPS

H SH HH HSSSS KKKKK

H H

HSKH

HSSKH

HS KKH

HindIII digestion

DNA CLONING: DNA LIGASE

TTCGA

5’

3’

A AGCTT 3’

5’A

TTCGA

5’

3’

A AGCTT 3’

5’A

DNA ligase+

ATP

-OH

-POH-

P-

2 ATP

2 AMP +2PPi

DNA CLONING: plasmid vectors

bacterial plasmid

origin of replication

(ori)

multiple cloning

site (MCS)- HindIII

- EcoRI

- KpnI

- SmaI

- BamHI

- XbaIampicillin resistance gene (amp)

E. coli

“TRANSFORMED”BACTERIA

“VECTOR”

DNA CLONING: TRANSFORMATION

E. coli

+

“COMPETENT CELLS”Chemically treated to enhance

DNA uptake

+

DNA CLONING: SELECTION

+

Luria Broth Agar+

Ampicillin

ONLY AMPICILLIN-RESISTANT (PLASMID-CONTAINING) BACTERIA CAN GROW

DNA CLONING: LARGE SCALE GROWTH

millions of copies of the recombinant

plasmid

PLASMID: A circular double-stranded DNA molecule that replicates in bacteria and is separate from the bacterial genome• engineered to contain only sequences needed to function as a DNA cloning vector:

• a bacterial origin of replication (ori)• an antibiotic resistance gene (eg. B-lactamase confers resistance to ampicillin (amp))• one or more unique restriction enzyme cutting sites which can be used to insert a piece of foreign DNA (MCS)

• may contain a B-galactosidase gene that is interrupted when DNA is inserted into the MCS• may also contain promoters that drive expression of a foreign gene in either prokaryotic or eukaryotic cells

DNA CLONING: PLASMIDS

Movie cloning

cDNAscDNAs

Clone LibrariesClone Libraries

Detection of specific RNA or DNA molecules Detection of specific RNA or DNA molecules by gel-transfer hybridizationby gel-transfer hybridization

slide 1slide 1

Detection of Detection of specific RNA or specific RNA or DNA molecules DNA molecules by gel-transfer by gel-transfer hybridizationhybridization

slide 2slide 2

DNA SequencingDNA Sequencing

Dideoxy-Sequencing (Sanger)Dideoxy-Sequencing (Sanger)

Dideoxy-Sequencing (Sanger) cont’dDideoxy-Sequencing (Sanger) cont’d

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Dideoxy-Sequencing (Sanger) Dideoxy-Sequencing (Sanger) cont’dcont’d

Reading Frames (6)Reading Frames (6)

Genes are found on either DNA Genes are found on either DNA strandstrand

Polymerase Chain Reaction Polymerase Chain Reaction (PCR) slide 1(PCR) slide 1

Polymerase Chain Reaction Polymerase Chain Reaction (PCR) slide 2(PCR) slide 2

Polymerase Chain Reaction Polymerase Chain Reaction (PCR) slide 3(PCR) slide 3

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PCR: Genomic or cDNAPCR: Genomic or cDNA

Technology allows you to move Technology allows you to move from protein to gene and from gene from protein to gene and from gene

to proteinto protein

Fusion Proteins for Analysis of FunctionFusion Proteins for Analysis of Function

Fluorescence Energy Transfer (FRET)Fluorescence Energy Transfer (FRET)

Affinity Coupled with Immunoprecipitation Tags Affinity Coupled with Immunoprecipitation Tags Facilitates the ID of Associated ProteinsFacilitates the ID of Associated Proteins

Yeast-Two-Hybrid Assay Yeast-Two-Hybrid Assay is used to discover protein-protein interactionsis used to discover protein-protein interactions

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To study the function of proteins To study the function of proteins in vivoin vivo one one needs to identify mutants within the gene needs to identify mutants within the gene

that encodes your protein and evaluate the that encodes your protein and evaluate the outcome.outcome.

Temperature Sensitive (TS) Mutants in Bacteria or Yeast

The use of TS-mutants in yeast identified The use of TS-mutants in yeast identified proteins that played critical roles in the proteins that played critical roles in the

export of proteinsexport of proteins

Mutations introduce a phenotypeMutations introduce a phenotype

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Single Nucleotide Polymorphisms (SNP) can be used in Single Nucleotide Polymorphisms (SNP) can be used in Linkage Analysis to identify genes or Chromosomal Linkage Analysis to identify genes or Chromosomal regions that are responsible for inherited disordersregions that are responsible for inherited disorders

DNA DNA Microarrays Microarrays monitor the monitor the

expression of expression of thousands of thousands of genes in one genes in one experimentexperiment

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Cluster Analysis used to identify sets of Cluster Analysis used to identify sets of genes that are coordinately regulatedgenes that are coordinately regulated

The expression of 8600 genes (Columns) were analyzed under 12 time points. Red represents increase in expression green a decrease relative to untreated cells.

Cells can be genetically engineered to Cells can be genetically engineered to carry different types of mutationscarry different types of mutations

Embryonic Embryonic Stem (ES) Stem (ES)

cells can be cells can be genetically genetically engineered engineered and used to and used to make a new make a new

animalanimal

The genetically The genetically engineered ES engineered ES

cells are used to cells are used to generate a generate a

chimeric animal, chimeric animal, which is then which is then used to make used to make

completely ES-completely ES-derived animalsderived animals