Unit 1: Basic Chemical and Biological Principals - PowerPoint Presentation

1. Unit 1: Basic Chemical and Biological Principals Chapter 4 - Genes, Genomes, and DNA

2. Figure 4.01. Neutrophil Extracellular Traps (NETs)Contact with bacteria, protozoa, yeast, or filamentous fungi (hyphae), or their products (e.g., bacterial lipopolysaccharide) stimulates neutrophils. This causes (A) the chromatin to unravel and the nuclear membrane to fragment, which allows cytoplasmic proteins to bind to the DNA. Next, (B) the DNA is released, together with bound histones and cytoplasmic proteins. This generates neutrophil extracellular traps (NETs) that ensnare and kill microorganisms.(Credit: Fig. 1 in Guimarães-Costa, A.B., Nascimento, M.T., Wardini, A.B., Pinto-da-Silva, L.H., Saraiva, E.M., 2012. ETosis: a microbicidal mechanism beyond cell death. J. Parasitol. Res. 2012, 929743.)

3. Figure 4.02. Symbiosis with Respiring Bacteria Gives Rise to the Primitive EukaryoteThe ancestor to the eukaryote, or “urkaryote,” engulfs a respiring bacterium by surrounding it with an infolding of the cell membrane. Consequently, there is now a double membrane around the newly enveloped bacterium. The symbiont, now called a “mitochondrion,” divides by fission like a bacterium and provides energy for the primitive eukaryote. The mitochondrion develops infoldings of the inner membrane that increase its energy producing capacity.

4. Figure 4.03. Genome Size of Organelles and SymbiontsThe mitochondria and chloroplasts are organelles that are derived from symbiotic prokaryotes. This plot shows genome size versus number of genes for free-living prokaryotes, present day endosymbionts and organelles. The term “plastid” includes chloroplasts and related organelles that have lost the ability to photosynthesize.(Credit: Fig. 1 in McCutcheon, J.P., 2016. From microbiology to cell biology: when an intracellular bacterium becomes part of its host cell. Curr. Opin. Cell Biol. 41, 132–136.)

5. Figure 4.04. Psyllid that Contains Carsonella SymbiontAdult hackberry petiole gall psyllid, Pachypsylla venusta, contains endosymbiotic bacteria, Carsonella ruddii, that have the smallest cellular genome known.(Credit: Photograph by Jerry F. Butler, UF/IFAS.)

6. Figure 4.05. Intervening Sequences Interrupt Eukaryotic GenesRegions of noncoding DNA between genes are called intergenic DNA. Noncoding regions that interrupt the coding regions of genes are called introns.

7. Figure 4.06. Deduction of Consensus SequenceThe frequency of base appearances is used to derive a consensus sequence that is most representative of the series of related sequences shown.

8. Figure 4.07. Structure of the LINE-1 ElementAn example of a Long interspersed elements (LINE-1) or L1 element is shown. L1 contains blocks of DNA that show homology with the pol and LTR sequences of retroviruses, as well as two coding sequences or open reading frames (ORF1 and ORF2) involved in its own replication.

9. Figure 4.08. Major Components of the Eukaryotic GenomeMajor types of sequence found in a typical eukaryotic genome. Tandem repeats are found in blocks whereas transposable elements and their derivatives form “dispersed repeats” so that individual copies are scattered around, more or less at random.

10. Figure 4.09. Density Gradient Centrifugation and Satellite BandsA cesium chloride gradient will reveal two (or more) bands of fragmented DNA if these differ in density. In this case, the lighter DNA contains sequences that are primarily satellite DNA.

11. Figure 4.10. Unequal Crossover Due to MisalignmentA pair of homologous chromosomes contains repeated elements. Since repeated elements may be readily misaligned during meiosis, crossing over will sometimes occur in regions that are not comparable in each chromosome. The result is one longer and one shorter DNA fragment.

12. Figure 4.11. Repeating Motifs in Mouse Satellite DNAVariations in the consensus 9 bp satellite DNA sequence GAAAAATGT are shown.

13. Figure 4.12. Palindromes and Inverted RepeatsA mirror-like palindrome and an inverted repeat are shown. Similar colors indicate palindromic or inverted sequences.

14. Figure 4.13. A HairpinIf a single strand of DNA containing inverted repeats is folded back upon itself, base pairing occurs forming a hairpin structure.

15. Figure 4.14. Stem and Loop MotifIf inverted repeats are separated by a few bases, a stem and loop structure results. The loop contains unpaired bases (NNN).

16. Figure 4.15. DNA Bending Due to Multiple A-tracts(A) Bending of DNA occurs to the 3′-side of A-tracts. (B) Such bending decreases the speed at which DNA travels during electrophoresis. Indeed, the mobility of a DNA molecule of a given length varies depending on the location of bent regions within the molecule. Bends in the middle have greater effect than those close to the ends.

17. Figure 4.16. Folding of a G-QuadruplexThe consensus sequence for G-quadruplex formation comprises four tracts of at least three guanines (red) separated by one to seven other bases. Quartets of four guanines are held together by noncanonical hydrogen bonds. These quartets are stacked in three dimensions to form the overall G-quadruplex. These structures may be parallel or antiparallel (as here).(Credit: Fig. 1 in Valton, A.L., Prioleau, M.N., 2016. G-quadruplexes in DNA replication: a problem or a necessity? Trends Genet. 32, 697–706.)

18. Figure 4.17. Supercoiling of DNABacterial DNA is negatively supercoiled in addition to the twisting imposed by the double helix.

19. Figure 4.18. Bacterial Chromosomes Loop From a Protein ScaffoldSupercoiling of bacterial DNA results in giant loops of supercoiled DNA extending from a central scaffold.

20. Figure 4.19. Structure of Condensin ComplexesProteins known as condensins help to maintain the looped structure of both bacterial and eukaryotic chromosomes. Structures shown are (A) from a typical bacterium, (B) from enteric bacteria including E. coli, (C) from a eukaryote. The color-coded labels show the different protein subunit terminology. Note that the prokaryotic condensin complexes possess identical SMC protein dimers (MukB in enteric bacteria) whereas the eukaryotes have a heterodimer of two similar subunits. The structure for E. coli is slightly different in arrangement, involving two complexes joined by MukF.(Credit: Fig. 1 in Palecek, J.J., Gruber, S., 2015. Kite proteins: a superfamily of SMC/Kleisin partners conserved across bacteria, archaea, and eukaryotes. Structure 23, 2183–2190. Elsevier.)

21. Figure 4.20. Mechanism of Type I and II TopoisomerasesThe difference in action between topoisomerases of Type I and Type II is in the breakage of strands. Type I breaks only one strand, while Type II breaks both strands. When one strand is broken, the other strand is passed through the break to undo one supercoil. When two strands are broken, double-stranded DNA is passed through the break and the supercoiling is reduced by two. After uncoiling, the breaks are rejoined.

22. Figure 4.21. Unlinking of Catenanes by TopoisomeraseTopoisomerases may uncoil, unknot, or unlink DNA as well as carry out the coiling, knotting, or interlinking of DNA. Topoisomerases act (at the locations shaded blue) by cutting both strands of the DNA at one location and passing another region of the DNA through the gap.

23. Figure 4.22. Cruciform Structure Formed from an Inverted RepeatBecause the DNA is palindromic, the strands can separate and base pair with themselves to form lateral cruciform extensions.

24. Figure 4.23. Principle of ElectrophoresisCreating an electrical field in a solution of positively and negatively charged ions allows the isolation of the ions with different charges. Since DNA has a negative charge due to its phosphate backbone, electrophoresis will isolate the negatively charged DNA from other components.

25. Figure 4.24. Agarose Gel Electrophoresis of DNAAgarose gel electrophoresis separates fragments of DNA by size. Negatively charged DNA molecules are attracted to the positive electrode. As the DNA migrates, the fragments of DNA are hindered by the cross-linked agarose meshwork. The smaller the piece of DNA, the less likely it will be slowed down. Therefore, smaller fragments of DNA migrate faster.

26. Figure 4.25. Agarose Gel Separation of DNA: Staining and StandardsTo visualize DNA, the agarose gel containing the separated DNA fragments is soaked in a solution of ethidium bromide, which intercalates between the base pairs of the DNA. Excess ethidium bromide is removed by rinsing in water, and the gel is placed under a UV light source. The UV light excites the ethidium bromide and causes it to fluoresce orange. In sample A, there are two fragments of DNA each of a different size and each forming a separate band in the gel. To determine the size of fragments, a standard set of DNA fragments of known sizes is run alongside the sample to be analyzed.

27. Figure 4.26. Separation of Supercoiled DNA by Electrophoresis(A) Supercoiled DNA molecules, all of identical sequence, were electrophoresed to reveal multiple bands, with each band differing in the number of supercoils, which is shown beside the band. Zero refers to relaxed or open circular DNA. (B) Actual ethidium bromide stained electrophoresis gels of four differing concentrations (A: 6.1 nM; B: 9.2 nM; C: 12.2 nM; D: 18.3 nM) of negatively supercoiled E. coli plasmid DNA (pXXZ06) incubated with pure DNA topoisomerase I. In each panel, lanes 1–7 contain DNA samples incubated at 37°C for 0, 0.5, 1, 2, 5, 10, and 15 min. Symbols: R=relaxed or open circular; S=supercoiled.(Credit: Xu, X, Leng, F, 2011. A rapid procedure to purify Escherichia coli DNA topoisomerase I. Prot. Exp. Purification 77, 214–219.)

28. Figure 4.27. Comparison of B-DNA, A-DNA, and Z-DNASeveral structurally different versions of the double helix exist. Shown here is the normal Watson-Crick double helix, the B-form (middle), together with the rarer A-form (left) and Z-DNA form (right).(Credit: Photo courtesy of Richard Wheeler; Wikicommons.)

29. Figure 4.28. Structure of H-DNAThis triple helix is formed by GA- and TC-rich regions of a plasmid and is composed of triads of bases.

30. Figure 4.29. Chromatin TerritoriesEach of the different colors represents a different chromosome from chicken situated in a separate chromatin territory. Since the chicken is diploid, there are two chromatin territories for each chromosome. The model proposed by this paper suggests that the chromatin territory is separated from another by an inter-chromatin space. This organization could partly explain how some eukaryotic genes are expressed and others are not expressed.(Credit: Fig. 2 in Cremer, T., Cremer C., 2001. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat. Rev Genetics 2, 292–301.)

31. Figure 4.30. Nucleosomes and HistonesThe basic unit in the folding of eukaryotic DNA is the nucleosome as shown here. A nucleosome is composed of eight histones comprising a core and one separate histone (H1) at the site where the wrapped DNA diverges. The enlarged region shows the packing of histones in the core. The H3-H4 tetramer dictates the shape of the core. Only one of the H2A and H2B dimers is shown; the other is on the other side, hidden from view.

32. Figure 4.31. Summary of the Folding of DNA in Eukaryotic ChromosomesThe DNA helix (A) is wrapped around (B) eight histones (the core). The linker DNA regions unite the nucleosomes to give a “string with beads.” This in turn is coiled helically (C) (not clearly indicated) to form a 30 nm fiber. The 30 nm fibers are further folded by looping and attachment to a protein scaffold. Finally, during mitosis the DNA is folded yet again to yield very thick chromosomes.

33. Figure 4.32. Histone H1 Links NucleosomesThe positioning of H1 (blue) above the DNA wrapped around the core particles allows one H1 to bind to another along a linear chain of nucleosomes. This helps in the tighter packing of the nucleosomes.

34. Figure 4.33. Histone Tails May be AcetylatedThe N-terminal domains of some of the histone proteins are free for acetylation as indicated by “acetyl.” The single letter system for naming amino acids is used.

35. Figure 4.34. Looping of 30 Nanometer Fiber on Chromosome Axis(A) A chain of nucleosomes is coiled further with six nucleosomes forming each turn. (B) The coiled nucleosomes form a helix, known as a 30 nm fiber. (C) The 30 nm fibers form loops that are periodically anchored to protein scaffolding.

36. Figure 4.35. Interphase and Metaphase ChromosomesBetween rounds of cell division, chromosomes consist of single chromatids and are referred to as interphase chromosomes. Before the next cell division, the DNA is replicated and each chromosome consists of two DNA molecules or chromatids linked at the centromere. Just prior to mitosis, condensation occurs, making the chromosomes (and chromatids) visible. The chromosomes are best viewed while spread out during the middle part (metaphase) of mitosis. Each daughter cell will acquire one of the chromatids and the process begins anew.