Download Chapter 7 Manipulating Proteins, DNA, and RNA

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