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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) 1Ab: 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