Chapter 24

1. Chapter 24 Genes, Genomes, and Chromosomes

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

3. Prokaryotic and Eukaryotic Genomes • 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.

4. Analysis of Genome complecisty by DNA reassociation C0 t1/2 is directly related to the amount of DNA in the genome (or called complexity)

5. Examples of DNA reassociation • Let’s assume the genome sizes of bacteriophage P and bacteria B are 103 and 106 bp, respectively. And the DNA sequences are unique (i.e., no repetitive sequences). • Imagine what would be the situation for Co of these two DNA? (say the average DNA fragment is 103 bp and a total of 106 bp DNA is present in the reaction mixture). • Phage DNA should contain 1000 copies of identicalDNA fragments, but bacterial DNA should contain 1000 copies of unique DNA fragments (none of them are the same).

6. The Cot1/2 is directly related to the complexity (total length of different sequences) in the genome.

7. Prokaryotic and Eukaryotic Genomes • 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.

8. Eukaryotic genomes have several sequence components • Repetitive DNA sequences in eukaryotic genomes include satellite DNAs and scattered duplicate sequences (LINES,SINES) • 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.

9. Eukaryotic genes are often interrupted • 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.

11. Numbers of Genes in Prokaryotic Genomes

12. Total gene number is known for several organisms

13. Determining Genome Nucleotide Sequences • Mapping genes by classical genetic recombination: • 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. • Recombination frequency between two genes in the same chromosome may be used to construct genetic map.

14. Physical Mapping • FISH ( fluorescent in situ hybridization) • Restriction maps • Southern blotting • REFP (restriction fragment length polymorphism) in DNA fingerprinting and locating genes • Cloning and DNA sequencing

15. Restriction and Modification • 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.

16. Restriction-Modification systems • Restriction enzymes of most use to biologists cleave both DNA strands site-specifically, depending on base methylation.

17. Type II restriction systems

18. Restriction Maps • Fragmentation of lbacteriophage DNA with restriction endonucleasesEcoRI or BamHI: • Experimental determination of fragmentation patterns, resulting from enzymatic digestion of the 48.5 kb linear DNA molecule from phage l. • 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. • Maps of cleavage sites for each enzyme on the DNA molecule.

19. Southern blotting for detection of specific nucleotide sequences • The principle of Southern transfer and hybridization: • Southern blotting permits detection of minute amounts of DNA in the presence of a vast excess of nonspecific DNA.

20. Analysis of a restriction fragment lengthpolymorphism:

21. DNA sequence variation in the human genome: • The T insertion in the 13th line is found at low frequency.

22. Cloning and DNA sequencing Sequencing a large chromosome by individual sequence determination of cloned fragments in contiguous sequences:

23. Genes and Chromosomes • What is gene? • One gene-one enzyme. • One gene-one protein (polypeptide). • Genes are segments of DNA that code for polypeptides and RNAs. • What is chromosome? Chromosome consists of one covalently connected DNA molecule and associated proteins • Viral genomic DNA may be associated with capsid proteins • Prokaryotic DNA is associated with proteins in the nucleoid • Eukaryotic DNA is organized with proteins into a complex called the chromatin

24. Packaging of DNA Packing ratio: the length of the DNA divided by the length of the unit that contains it.

25. DNA from a lysed E. coli cell

26. Structure of a bacterial nucleoid 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 mm in length. An alternative form of negative supercoiling, solenoidal, allows greater compaction and is seen in chromatin.

27. The Nucleus, Chromosomes, and Chromatin

28. Metaphase chromosome • A mitotic chromosome: • An electron microscope image of a human chromosome during the metaphase stage of mitosis. • The constriction at the centromere and the lengthwise division into sister chromatids are clearly visible. • The hairy-looking surface is made of loops of highly coiled chromatin. • The chromatin of eukaryotes consists of DNA complexed with histones and nonhistone proteins.

29. Centromeres and Telomeres Mitotic human chromosomes in metaphase, stained separately by FISH for centromeres (pink) and telomeres (green):

30. How is DNA packed in the chromosomes • DNA in the cell must be organized to allow: • Packing of large DNA molecules within the cells • Access of proteins to read the information in DNA sequence • DNA Supercoiling. • Proteins assisted packaging (nucleosomes)

31. Supercoiling of DNA can only occur in closed-circular DNA or linear DNA where the ends are fixed. Underwinding produces negative supercoils, wheres overwinding produces positive supercoils.

32. Negative and positive supercoils . Topoisomerases catalyze changes in the linking number of DNA.

33. Topology of cccDNA is defined by: Lk = Tw + Wr, where Lk is the linking number, Tw is twist and Wr is writhe.

34. DNA Compaction Requires Solenoidal Supercoiling, not plectonemic supercoiling. Types of supercoiling found in chromosomes:

35. Protein-assisted Packaging of DNA Nucleosomes are the fundamental organizational units of eukaryotic chromatin

36. Nucleosomes in extended chromatin fiber • The elements of chromatin structure: • At the top is our current understanding of the extended structure of a chromatin fiber. • Light digestion with nuclease releases first mononucleosomes and oligonucleosomes. • Then, as linker DNA is further digested, nonhistone proteins and H1 are released, to yield the core particle

37. Histone octamer core of nucleosome • The basic repeating structure in chromatin is the nucleosome, in which nearly two turns of DNA are wrapped about an octamer of histones.

38. Loops of DNA Attached to a Chromosomal Scaffold

39. Matrix Attachment Regions (MAR) • Attachment of gene clusters to the nuclear matrix: • A map of the repeating histone gene cluster in Drosophila, where each white arrow is a histone gene (arrowheads indicate direction of transcription). • If Drosophila nuclei are extracted with lithium diiodosalicylate, to remove proteins gently, and are then digested with a collection of the restriction endonucleases shown, only the 657-bp HindI-EcoRIDNA fragments are left attached to the matrix. • The interpretation is that the gene clusters exist in individual loops, the bases of which are tied to the matrix.

40. Nucleosomes are packed into successively higher-order structures • Levels of chromatin structure: • To the left is a schematic view of a portion of the nucleus, with partially condensed chromatin fibers. • A closer view (to the right) shows a chromatin fiber in which part is in the condensed (30 nm) form, and part is opened up, as for transcription.

41. The Cell Cycle • The eukaryotic cell cycle: • Changes in the amount of DNA (blue line) and rate of histone synthesis (red line) with time during two cell cycles. • The DNA content is measured in units of the haploid genome (C). • The time scale is typical of many eukaryotic cells.

42. Mitosis • Mitosis: • The cell entering the pathway was originally diploid. • It has undergone DNA replication and is now in G2, with a DNA content of 4C. • After the process is complete, each daughter cell will again be 2C.

43. Structural organization of chromatinin the human centromere • Interaction between the outer region of each of the paired chromatids with kinetochores and microtubules is schematized as well. • H3-K9 and H3-K27 refer to histone modifications—methylation of lysine 9 and 27, respectively, on histone H3.

44. A model for kinetochore structure and organization: • The model is based upon electron microscopic and proteomic analysis of kinetochores in yeast (S. cerevisiae). • Cse4 and CENP-A are alternative names for the modified nucleosomes in the centromere.

45. Control of the cell cycle by cyclin-dependent kinases, and the checkpoints • The major checkpoints, showing the conditions checked and the regulatory proteins involved at each checkpoint. • The pattern of synthesis and degradation of each cyclin throughout the cell cycle. • Activities of the cyclin-dependent kinases remain constant.

46. Polymerase Chain Reaction • 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.

47. Polymerase Chain Reaction • 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.