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Copyright © 2013 Pearson Canada Inc. 24 - 1 Chapter 24 Genes, Genomes, and Chromosomes
Biochemistry, 4 th Edition Copyright © 2013 Pearson Canada Inc. 24 - 2 Chapter 24 Outline: Prokaryotic and Eukaryotic Genomes Restriction and Modification Determining Genome Nucleotide Sequences Physical Organization of Genes: The Nucleus, Chromosomes, and Chromatin The Cell Cycle Polymerase Chain Reaction I expect that you can draw all the bases, nucleoside and nucleotides of DNA and RNA and that you can draw a short segment of a polymer. In other words, You need to remember what you learned in Matthews Chapter 4!!!
Biochemistry, 4 th Edition The central dogma of molecular biology.
Biochemistry, 4 th Edition Prokaryotic and Eukaryotic Genomes Copyright © 2013 Pearson Canada Inc. 24 - 4 Genome size: The bars show the range of haploid genome sizes for different groups of organisms. A few specific organisms are marked with vertical lines, for example for humans. Note that the genomesize scale is logarithmic and that many organisms have larger genomes than humans.
Biochemistry, 4 th Edition Prokaryotic and Eukaryotic Genomes Copyright © 2013 Pearson Canada Inc. 24 - 5 Reassociation kinetics of E. coli and bovine DNA: The abscissa corresponds to reassociation time, corrected for the difference in size between the E. coli and bovine genomes. The curve for E. coli corresponds to that expected for a collection of single-copy genes in a genome of the E. coli size— 6 bp. The curve for bovine DNA exhibits two steps in reassociation. The slow step corresponds to single-copy DNA (nonrepeated sequences). The other corresponds to rapidly reassociating DNA made up of repeated sequences. Many classes of repeated DNA are represented in this phase of the reassociation.
Biochemistry, 4 th Edition Prokaryotic and Eukaryotic Genomes Copyright © 2013 Pearson Canada Inc. 24 - 6 Repetitive DNA sequences in eukaryotic genomes include satellite DNAs and scattered duplicate sequences. Satellite DNA: Equilibrium density gradient centrifugation of total Drosophila DNA resolves satellite bands, surrounding the main band. These represent repetitive DNA fractions of differing base composition.
Biochemistry, 4 th Edition Prokaryotic and Eukaryotic Genomes Copyright © 2013 Pearson Canada Inc. 24 - 7 Exon–intron structure of the ovalbumin gene in chickens: Map of the 7700 bp gene, showing exons 1–7 plus an untranslated leader sequence (blue) and introns A–G (brown). Electron micrograph of a hybrid formed by renaturing chicken genomic DNA with purified ovalbumin mRNA. Diagram showing how the intron regions loop out in R loops in such a hybrid. The RNA is shown in red, the DNA exons in blue, and the DNA introns in brown.
Biochemistry, 4 th Edition Prokaryotic and Eukaryotic Genomes Copyright © 2013 Pearson Canada Inc. 24 - 8
Biochemistry, 4 th Edition Restriction and Modification Copyright © 2013 Pearson Canada Inc. 24 - 9 Bacteria use restriction–modification, which involves nongenetic changes in DNA structure, to distinguish their own DNA from that of invaders. Host-induced restriction and modification: A phage whose DNA is unmodified infects a bacterium with a restriction system that recognizes the DNA sequence 5’– GAATTC–3’ (step 1). Most phage DNA molecules are cleaved by the restriction nuclease (step 2), but the few that become methylated first on the innermost A are protected from attack (step 3). The phages that emerge contain modified (methylated) DNA (step 4). Because they are not vulnerable to restriction by the host nuclease, they are able to overcome the bacterium’s defense system when they reinfect the same bacterial strain.
Biochemistry, 4 th Edition Restriction and Modification Copyright © 2013 Pearson Canada Inc. 24 - 10 Fragmentation of bacteriophage DNA with restriction endonucleases EcoRI or BamHI: a)Experimental determination of fragmentation patterns, resulting from enzymatic digestion of the 48.5 kb linear DNA molecule from phage. Restriction digests are subjected to agarose gel electrophoresis, and the fragments are visualized under ultraviolet light after staining the gel with ethidium bromide. Note that fragments with very similar sizes form but one band on a gel. b)Maps of cleavage sites for each enzyme on the DNA molecule.
Biochemistry, 4 th Edition Restriction and Modification Copyright © 2013 Pearson Canada Inc. 24 - 11 Restriction enzymes of most use to biologists cleave both DNA strands site-specifically, depending on base methylation.
Biochemistry, 4 th Edition Restriction and Modification Copyright © 2013 Pearson Canada Inc. 24 - 12
Biochemistry, 4 th Edition Restriction and Modification Copyright © 2013 Pearson Canada Inc. 24 - 13 Structure of the EcoRI nuclease complexed with its DNA substrate: The DNA helix is shown in blue, while the two subunits of the protein are shown in red and yellow, respectively. Note the “kink” in the DNA structure, resulting from the fact that the enzyme binds the central six-base-pair cutting site in the B conformation, while the flanking sequences are bound as A-form DNA. Note also the N-terminal “arm” on each protein subunit, which wraps around the DNA.
Biochemistry, 4 th Edition Restriction and Modification Copyright © 2013 Pearson Canada Inc. 24 - 14 Structures of (a) free and (b) DNA-bound forms of BamHI, with DNA shown end-on, in orange: Regions of the protein that undergo conformational change upon DNA binding, including the two C-terminal helices, are shown in yellow.
Biochemistry, 4 th Edition Determining Genome Nucleotide Sequences Copyright © 2013 Pearson Canada Inc. 24 - 15 Structure of a complex of a type II DNA methylase with DNA: The structure is a ternary complex containing Hha methylase from Haemophilus haemolyticus, DNA, and S-adenosylhomocysteine. The loops containing the catalytic site are in white, and the rest of the enzyme is in orange. S-Adenosylhomocysteine is in yellow, the DNA backbone is magenta, and bases are green. In both views the flipped-out target cytosine base is clearly visible. a)View looking down the helix. b)Side view from the minor groove.
Biochemistry, 4 th Edition Determining Genome Nucleotide Sequences Copyright © 2013 Pearson Canada Inc. 24 - 16 Segregation of two genetic markers, lying either on separate chromosomes (top) or linked on the same chromosome (bottom): A and B are dominant alleles of genes in which the genotype can be inferred from direct observation of a phenotype, such as eye color or wing shape. In each case, two heterozygous parents are mated, and the expected proportion of each genotype in the progeny is shown. When the genes being analyzed lie on different chromosomes, the markers assort randomly. When they lie on the same chromosome, wild-type (AaBb) or double-mutant (aabb) progeny arise only through relatively rare recombination events.
Biochemistry, 4 th Edition Physical Organization of Genes: The Nucleus, Chromosomes, and Chromatin Copyright © 2013 Pearson Canada Inc. 24 - 17 Types of supercoiling found in chromosomes:
Biochemistry, 4 th Edition Physical Organization of Genes: The Nucleus, Chromosomes, and Chromatin Copyright © 2013 Pearson Canada Inc. 24 - 18 Structure of a bacterial nucleoid, showing independent domains of supercoiling, each stabilized by binding to protein: The term plectonemic refers to the type of supercoiling observed, with DNA strands intertwined in a regular way. The 1000 nm diameter of the structure allows it to fit within a bacterial cell that may be 2 to 5 m in length. An alternative form of negative supercoiling, solenoidal, allows greater compaction and is seen in chromatin.
Biochemistry, 4 th Edition Polymerase Chain Reaction Copyright © 2013 Pearson Canada Inc. 24 - 19 The polymerase chain reaction (PCR), was invented by Kary Mullis in 1983. PCR allows the amplification of exceedingly small amounts of DNA in vitro, without prior transfer into living cells. To understand PCR, you must understand that DNA polymerase catalyzes the addition of deoxyribonucleoside triphosphate to a pre-existing 3’-hydroxyl terminus of a growing daughter DNA strand (the primer). A template DNA strand is required, to instruct the polymerase regarding the correct nucleotide to insert at each step.
Biochemistry, 4 th Edition Polymerase Chain Reaction Copyright © 2013 Pearson Canada Inc. 24 - 20 Three cycles of PCR: A segment within the region shown in blue is amplified, by use of primers (red) that are complementary to the ends of the blue segment. Note the exponential nature of the amplification process.
Biochemistry, 4 th Edition A little history…. What was the Meselsohn and Stahl experiment? Avery? And now for Hershey and Chase...
Biochemistry, 4 th Edition Bacteriophages attached to the surface of a bacterium. Page 84
Biochemistry, 4 th Edition Figure 5-8The Hershey-Chase experiment. Page 85 1952 Alfred Hershey and Martha Chase
Biochemistry, 4 th Edition Diagram of T2 bacteriophage injecting its DNA into an E. coli cell. Page 84
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