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week%209-14 - Two-Dimensional Gel Electrophoresis • First...

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Unformatted text preview: Two-Dimensional Gel Electrophoresis • First, isoelectric focusing. • Then standard SDS-PAGE. Immunoblotting (Western blotting) for specific protein detection • • • The antibody that recognizes the protein of interest (the primary antibody, 1o) is incubated with the blot. Next, the blot is incubated with an antibody (secondary, 2o) that recognizes the constant fragment (Fc) of the 1o antibody. The 2o antibody is attached to an enzyme that produces a chromogenic or chemiluminescent reaction in the presence of the appropriate substrate. The quaternary structure of Immunoglobulin G IgG specificity is determined by its variable regions Fig. 4-10 The variation occurs through DNA rearrangements in antibody producing immune cells (B cells). Antibodies are also important research tools • specifically identify a protein in a very complex mixture (e.g.: Western blotting). • localize proteins in a cell (e.g.: Immunofluorescence) • protein purification (e.g.: chromatography). • Can be used diagnostically (e.g.: HIV). • Can be used as inhibitors of protein activity (in research and therapeutically). Direct sequencing of a protein--Edman degradation The structure of DNA and RNA Outline • Structure and topology of DNA • Structure of RNA • DNA analysis techniques Reading assignments: Molecular Biology of the Gene (Watson, 6th ed) chapter 6, chapter 21 p740-750 The DNA bases are purines and pyrimidines Formation of the nucleotides in DNA • Condensation reactions join deoxyribose, phosphoric acid, and a base to form each deoxynucleoside monophosphate. Compatible and Incompatible Base-Pairs • H-bond donors and H-bond acceptors must be properly positioned for compatible base-pairing. The Structure of DNA • The distances between the two DNA backbone strands is maintained constant. • The two DNA strands are in an antiparallel orientation. • The 5’ end has a phosphate group and the 3’ end has a hydroxyl. Different Forms of DNA helix • The B form (or something very similar to it) is the most common form in the cell. • The A- and B-forms are right-handed helices, while the Z-form is a lefthanded helix. • Z-DNA is favored by alternating G and C sequence, and unwound DNA. The Structure of the DNA Double Helix • Two anti-parallele strands. • About 10 bases per turn of the helix in the most common form of DNA • H-bonding and hydrophobic (basestacking) interactions hold the two strands together. The Structure of the DNA Double Helix • There is a major and minor groove on the surface of the helix that is an important site of interaction for many regulatory proteins. DNA sequence information in the major and minor grooves • In the major groove, all four base-pairings can be distinguished. • In the minor groove, only two of the four basepairings are clearly distinguished. Denaturation and Renaturation of DNA • Annealing of longer complement ary stretches of DNA will be favored over shorter ones. Hyperchromicity of single-stranded DNA Tm (melting temperature): the temperature at which half the DNA is denatured. Tm is determined by base composition and ionic strength • The higher the %G+C in the DNA molecule, the higher the Tm will be. • At higher ionic strength (e.g.: salt concentration) the Tm is higher, because ions shield the negative charges of the DNA backbone. low salt high salt RNA Structure • RNA is usually singlestranded (ss), • RNA has the 2’-OH, • RNA has uracil instead of thymine. • RNA is unstable at high pH because the 2’ -OH can react with the phosphate group. RNA forms double-stranded helices through base complementarity • When doublestranded, RNA adopts the Aform helix. Unusual structures formed by RNA molecules • G-U base-pairings occur (important for tRNA) • Base-backbone interactions occur Unusual Base-Pairing in RNA Facilitates the Formation of ds RNA Regions RNA can act as enzymes---ribozymes • The 2’ -OH allows RNA to catalyze reactions. Ribozyme self-cleavage: “Did Life Evolve from an RNA World?” The Central Dogma: DNA RNA Protein However, evolution of this system would have required two, concurrent evolutionary steps. The ability of RNA to act as both and information molecule and catalyst suggests RNA came first. Hence: DNA Later RNA First Second Protein Topology of Circular DNA Molecules • Supercoils are twists of the double helix above and beyond the normal turns of the double helix, can be either positive (overwinding) or negative (underwinding). • DNA in cells is negatively supercoiled. Topology of Circular DNA Molecules • Linking # is the # of turns of one strand around the other necessary to separate the two molecules • Twist is the # of complete helical turns of the two DNA strands • Writhe is the # of folds or super-helical turns of the ds helix • Linking # (Lk) = Twist (Tw) + Writhe (Wr) Relaxing DNA with DNase I • DNase I (deoxyribonuclease I) is an enzyme that hydrolyzes (cleaves) one strand of the DNA backbone nonspecifically . Topological Changes of DNA Are Carried Out by Topoisomerases Topoisomerases use covalent proteinDNA linkage Topoisomerase I makes singlestranded breaks in DNA • It will relax either positive or negative supercoiling. • Does not require energy (ATP) for the process. Mechanism of Topoisomerase I • Topo I cleaves one DNA strand and holds one end. • Rotation occurs around the phosphodiester bond of the uncleaved strand, relieving tension (pos or neg). • Topo I ligates the two DNA ends. Topoisomerase II makes doublestranded breaks in DNA • Tangles and knot-like structures are removed by Type II Topoisomerase. • Energy (ATP) is required. Mechanisms of Topoisomerase II • Topo II separates two tangled helices by cutting both strands of one helix and passing the other helix through the gap. • Topo II then ligates the two ends. • ATP is required. DNA topoisomers can be separated by electrophoresis • Agarose or polyacrylamide matrices are used to separate DNA molecules. Intercalation of Ethidium into DNA • Intercalation is the insertion of a compound between the bases. • Ethidium fluoresces in the presence of UV light, and is used to visualize DNA in a gel. Gel Electrophoresis of DNA Molecules Restriction endonucleases are sequencespecific DNA-binding proteins • Restriction enzymes bind DNA as dimers. • They recognize palindromic DNA sequences (two halfsites). • Cleave DNA symmetrically on both strands of the DNA. Restriction-modification systems protect the host genome from foreign DNA Restriction endonuclease digestion of DNA • The frequency of DNA cutting by a restriction endonuclease depends on the length of the DNA sequence recognized. • the frequency of digestion = (1/4)n where n = length of recognized sequence DNA blot hybridization (Southern Blot) • DNA is cut with a restriction enzyme, separated by gel electrophoresis, and blotted to a membrane. • A DNA “probe” with a specific sequence can be used to identify any complementary DNA molecules on the blot. • The probe is usually radioactive or otherwise labeled for detection. • This method can be used to identify differences in DNA between individuals. End-labeling a DNA molecule P HO OH P Treat with phosphatase HO HO OH OH Treat with kinase plus -32P-ATP P HO OH P Optional: for a probe with a single labeled end, remove one end with a restriction enzyme P HO OH P EcoRI “Cloning” DNA • A plasmid vector requires: – a bacterial replication origin (to replicate the plasmid in the E. coli host) – a selectable marker gene (to select for the transformed cells) – a (multiple) cloning site (to insert the DNA of interest) Construction of a Genomic DNA library • A DNA library is a collection of different DNA fragments representing the entire genome of interest individually inserted into a plasmid (or other vector). • Each clone is equally represented (theoretically) • The DNA library is propagated in E. coli Synthesis of cDNA for a cDNA “Expression” library • Messenger RNA (mRNA) is unique in having a poly-A tail, which can be used to isolate mRNA from other forms of RNA. Genome structure and chromosome Outline • Genome structure • Chromosome duplication and segregation Reading assignments: Molecular Biology of the Gene (Watson, 6th ed) chapter 7 p135-157 Chromosomes of prokaryotic and eukaryotic cells • Chromosomes are compacted and organized proteinDNA structures (one DNA molecule). • Chromosomes can be circular or linear. • Haploid genomes (prokaryotes and eukaryotic gametes) have one copy of each chromosome; diploids have two copies of each chromosome. Euchromatin vs. heterochromatin • Heterochromatin: condensed, darkly stained, limited gene expression • Euchromatin: more open, lightly stained, high levels of gene expression Comparison of gene densities of organisms • Genomes are composed of genes and intergenic DNA. • Gene density decreases dramatically in the genomes of more complex organisms. • Intergenic regions contain pseudogenes and other genetic “garbage” and repetitive DNA sequences. Pseudogenes and repetitive DNA elements arise from reverse transcription • Reverse transcription is the copying of an RNA sequence into a DNA sequence. Eukaryotic genes may contain introns • Transcription produces a primary transcript that is processed by splicing and other modifications to produce a functional mRNA, which is transported to the cytoplasm and translated into protein. DNA elements required for chromosomal duplication, segregation, and maintenance • • • The Centromere is the assembly site of the Kinetochore (a proteinDNA complex) where the spindle attaches to segregate the chromosomes during mitosis and meiosis. Replication origins are the sites where DNA replication proteins assemble and initiate DNA replication. Telomeres are the ends of linear chromosomes, and serve as assembly sites for proteins that protect these ends from degradation and fusion with other DNA ends, and help in the replication of the ends of the DNA. Fewer or more than one centromere is highly detrimental Differences in centromeres of eukaryotes The eukaryotic mitotic cell cycle • During the Gap phases (G1 and G2) checkpoints ensure that all is well with the DNA and segregation machinery, before allowing DNA replication (S phase) and mitosis (M) to commence. Chromatin structure changes during the cell cycle decondensed condensed Various levels of chromatin compaction during cell cycle Chromosome Cohesion and Condensation by SMC Proteins • SMC proteins: the Structural Maintenance of Chromosomes proteins S Phase • Individual replication origins initiate replication and produce two replication forks per origin. • During replication cohesin proteins assemble around the new DNA molecules, holding them together (cohesion). • The fully replicated DNA molecules are called sister chromatids. M Phase • Sister chromatids separate by the end of M pahse. Mitosis • • Sister chromatids are segregated to daughter cells. Checkpoints regulate various steps, especially the metaphase to anaphase transition to ensure accuracy. Meiosis • After DNA replication, but before chromosome segregation, homologous (maternal and paternal) chromosomes pair and recombine. Two rounds of chromosome segregation follow. In meiosis I, homologous chromosomes segregate. In meiosis II, sister chromatids segregate (just like in mitosis). • • • ...
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