Chapter 7 Manipulating Proteins, DNA, and RNA

A fluorescence-activated cell sorter

Microdissection techniques to select cells from tissue slices

Embryonic stem cells (ES) derived from an embryo

Reproductive and therapeutic cloning

The production of hybrid cells

(a combined cell with two separate nuclei)

Preparation of hybridomas that secrete monoclonal antibodies against a particular antigen

The preparative ultracentrifuge

(Reduces friction)

Cell fractionation by centrifugation

1,000g → 10 min

20,000g → 20 min

80,000g → 1 hr

150,000g → 3 hr

Comparison of velocity sedimentation and equilibrium sedimentation

The separation of molecules by column chromatography

Cellulose

Three types of matrices used for chromatography Cross-linked polysaccharide - dextran - agarose - acrylamide

DEAE-cellulose (+) CM-cellulose (-) Phosphocellulose (-)

500 Da - 5000 kDa

Antibody Enzyme substrate

Cyclin B3 affinity column

Cyclin B2 affinity column

Total cell extract

Protein purification by chromatography

Epitope tagging for the localization or purification of proteins

Purification of protein complexes by using a GST-tagged fusion protein

The detergent sodium dodecyl sulfate (SDS) and the reducing agent -mercaptoethanol

SDS polyacrylamide-gel electrophoresis (SDS-PAGE)

To determine the approximate molecular weight

Complex mixture of proteins

Analysis of protein samples by SDS polyacrylamide-gel electrophoresis Chromatographic fractionation

Commassie-stained gel

Western blotting (Immunoblotting)

1Ab: anti-phosphothreonine

Polyacrylamide-gel

Nitrocellulose membrane

Use of spectrometry to identify proteins and to sequence peptides Lysine Arginine

Separation of protein molecules by isoelectric focusing -NH3+ -COOH

-NH2 -COO-

Two-dimensional polyacrylamide-gel electrophoresis

Radioisotope-labeled amino acids → autoradiography

The yeast two-hybrid system for detecting protein-protein interactions

Transcriptional activator

Surface plasmon resonance

Fluorescence resonance energy transfer (FRET)

Small-molecule inhibitors for manipulating living cells Microtubules

Control

(Kinesin inhibitor)

Chromosomes

Mitotic spindle Monastrol

X-ray crystallography

Ribose biphosphate carboxylase

Atomic model

NMR spectroscopy

2-D NMR spectrum

C-terminal domain of cellulase

The higher the alignment score the better the match identical

Results of a BLAST search The lower the E value the more significant the match

Conserved amino acids

similar identical

difference

Similarities

BLAST (Basic Local Alignment Search Tool)

The DNA nucleotide sequences recognized by four widely used restriction nucelases Recognition enzymes (from bacteria) Recognition sequence

Blunt ends

Cohesive ends (Sticking ends)

Palindromic sequence

A palindrome site is a sequence of base pairs in double stranded DNA that reads the same backwards and forward across the double strand.

The use of restriction nucleases to produce DNA fragments that can be easily joined together EcoR I

Gel electrophoresis techniques for separating GATC DNA molecules by size Autoradiography Ethidium bromide staining (A/T base pairs)

Polyacrylamide gel for single-strand DNA 10-500 nucleotides

Agarose gel for double-strand DNA 300-10000 nucleotide pairs

Ethidium bromide staining (A/T base pairs)

Pulse-field agarose gel for double-strand DNA 220,000-2,500,000 nucleotide pairs

Methods for labeling DNA molecules in vitro (1)

A single 5’-end-labeled strand

Methods for labeling DNA molecules in vitro (2)

Anti-digoxigenin antibody (Fluorescent dye labeled)

Modified Thymine

In situ hybridization to locate specific genes on chromosomes DNA probes

Fluorescent antibodies

Human chromosome 5 at metaphase

Stringent versus nonstringent hybridization conditions

Melting temperature (Tm)

Identical

Related

Stringent hybridization

Nonstringent hybridization

The use of nucleic acid hybridization to determine the region of a cloned DNA fragment that is present in an mRNA molecule

The insertion of a DNA fragment into a bacterial plasmid with the enzyme DNA ligase

The amplification of the DNA fragments inserted into a plasmids

Transformation

The making of a yeast artificial chromosome (YAC) Centromere

Origin of replication

Telomere

Very large DNA molecules

Construction of a human genomic DNA library

The synthesis of cDNA

DNA/RNA hybrid helix

The differences between cDNA clones and genomics DNA clones derived from the same region of DNA

Only part of the coding sequence of a gene

Transcribed DNA (Introns & Exons) Nontranscribed DNA

Exons only

The Nobel Prize in Chemistry 1993 “The Developments of methods within DNA-based chemistry”

The polymerase chain reaction (PCR) method

The establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies".

Amplification of DNA by the PCR technique

21=2

22=4

23=8

Use of PCR to obtain a genomic or cDNA clone

How PCR is used in forensic science

CACACA… 4-40 repeats VNTR (variable number of tandem repeat) = hypervariable microsatellite sequence

Production of large amount of a protein from a protein-coding DNA sequence cloned into an expression vector and introduced into cells

High active promoter

Bacterial cells Yeast cells Insect cells Mammalian cells

Production of large amounts of a protein by using a plasmid expression vector 0.5

1

2 hr

The enzymatic-or dideoxy-method of sequencing DNA

Radioactive or fluorescent labeled primer or dNTP

Automated DNA sequencing

Finding the regions in a DNA sequence that encode a protein Stop codon

1700 base-pair DNA

Screening for temperature-sensitive bacterial or yeast mutants

Gene mutations that affect their protein production in different ways

Using genetics to determine the order of function of genes

The gene defective in mutant A acts before the gene defective in mutant B in the secretory pathway

SNPs (Single-nucleotide polymorphisms) SNPs are single-nucleotide substitutions of one base for another that occur in more than one percent of the general population

Genetic linkage analysis using physical markers on DNA to find a human gene

Tracing the inheritance of SNPs within haplotype blocks to reveal the location of a disease-causing gene

Identification of alleles that have been selected for in fairly recent human history by the unusually large haplotype blocks in which they are embedded

Relatively recently in human history

Dominant-negative mutation

A dominant-negative effect of a protein

Site-directed mutagenesis

The use of a synthetic oligonucleotide to modify the protein -encoding region of a gene by site-directed mutagenesis

A single mismatched nucleotide pair

Gene replacement, gene knockout, and gene deletion

Such as dominant-negative mutation

Summary of the procedures used for making gene replacements in mice Embryonic stem cells

Transgenic mice engineered to express a mutant DNA helicase show premature aging DNA helicase (Xpd gene): transcription and DNA repair

Wild-type Xpd

Mutant Xpd Accumulation of DNA damage Symptoms of premature aging (1) Osteoporosis (2) Emaciation (3) Early aging (4) Infertility (5) Reduced life-span

A procedure used to make a transgenic plant

Kanamycin-resistance gene

T-DNA (transferred DNA)

Making collections of mutant organisms  20 nucleotide pairs

To prepare tagged knockout mutants

Dominant-negative mutation created by RNA interference

Wild-type

Mutant

Pronuclei from sperm and egg

Using a reporter protein to determine the pattern of a gene’s expression

Green fluorescent protein (GFP) -galactosidase (-gal)

Experiment demonstrating the modular construction of the Eve gene regulatory region

PCR (Polymerase Chain Reaction)

Complementary DNA (cDNA)

Semi-quantitative RT-PCR (RT-PCR)

RNA levels can be measured by quantitative RT-PCR

Quantitative RT-PCR (Q-PCR)

Using DNA microarrays to monitor the expression of Thousands of genes stimulatory

Using cluster analysis to identify sets of genes that are coordinately regulated Serum starvation for 48 hrs

Increase in expression

Decrease in expression

Serum added

Different levels of gene expression in individual cells within a population of E. coli bacteria

Two different reporter proteins (GFP, RFP) controlled by a copy of the same promoter