![Transcription factories in a Hela cell [from Cook PR (1999) Science 284, 1790]](../images/pombo.png)
Go to the same questions without answers.
Overview of the cell nucleus
1.Roughly, when were cells, nuclei, chromosomes, and DNA discovered? [1665, 1883, 1848, 1871.]
2.When did Mendel, and Watson and Crick publish their seminal works? [1865, 1953.]
3.Assuming a nucleus is 10 μm in diameter, how much smaller in diameter is a water molecule, a typical globular protein of 50 MDa, a nucleosome, a ribosome, and a nuclear pore? [3x10-5 times, 5x10-4 times, 10-3 times, 5x10-2 times, 10-2 times.]
4.What is the approximate contour length of DNA of a typical human chromosome containing 100 Mbp? [34 mm.] What is (roughly) the length of a mitotic chromosome? [3 μm.]
5.What is meant by the resolution of a microscope, and what is the resolution of the light and electron microscope? [Resolution: the minimum distance (D) that enables two objects to be distinguished, where D = 0.61λ/(Nsinα). LM: 200 nm at best. EM: 0.1 nm at best.]
6.What processes are involved in preparing an image like that of the histological section shown in Fig. 1-1? [Describe: fixation, embedding, sectioning, staining, and imaging. See next answer.]
7.What is meant by the terms fixation, embedding, and staining (as applied to a histological section like that illustrated in Fig. 1-1)? [Fixation: cross-linking so macromolecules are locked in position. Embedding: immersion in a supporting medium to permit sectioning. Staining: attaching stains so different regions appear different.]
8.What are the differences between freeze-fracture, freeze-drying, freeze-etching, and freeze-substitution? [Freeze-fracture: specimen is frozen, then fractured before a replica of the fractured surface is observed. Freeze-drying: specimen is frozen, then lyophilized before the remaining dry material is stabilized for direct observation, or preparation of a replica. Freeze-etching: combination of both freeze-fracture and freeze-drying. Freeze-substitution: specimen is frozen, then the ice is dissolved and replaced at low temperature by an organic solvent like acetone before embedding in plastic.]
9.What is the green fluorescent protein, and how does it fluoresce? [An autofluorescent protein found in the jellyfish, Aequoria victoria, responsible for the bioluminescence seen in a ship's wake. When calcium-ion levels change, aequorin is induced to emit blue light; this is transferred to GFP, which then emits green light. A chromophore of three amino acids (ser65-tyr66-gly67) undergoes sequential cyclization, loss of water, and oxidation during excitation and emission.]
10.Describe an experiment that makes use of the green fluorescent protein in cell biology.
11.Roughly, how long does it take (i) a typical protein to diffuse across a nucleus, and (ii) a segment of a human chromosome to diffuse 200 nm within a nucleus? [(i): 10-2 s. (ii): 30 s.] How was the data obtained? [Using dextrans tagged with fluorescein, or chromatin fibers tagged with the green fluorescent protein.]
12.Roughly, how far apart are molecules when in solution at 1 μM (and 1 mM)? [120 (and 12) nm.]
Structures of nucleic acids
13.What are the components of a nucleotide? [A purine or pyrimidine base, a 5-carbon cyclic sugar (linked to the base through a nitrogen), and a phosphate (esterified to the hydroxyl on the 5' carbon of the sugar).]
14.Draw the structure of a named nucleotide.
15.What are the chemical differences between DNA and RNA? [In RNA, U replaces T, and the sugar is ribose instead of deoxyribose.]
16.What are the purines and pyrimidines found in DNA and RNA? [Purines: A and G in both DNA and RNA. Pyrimidines: T and C in DNA, U and C in RNA.]
17.Draw the structures of A:T and G:C base pairs.
18.Draw the structure of the phosphodiester backbone in a single strand of DNA.
19.Is the double helix right or left handed? [Right-handed.]
20.What do we mean when we say that the strands in a DNA duplex are 'anti-parallel'? [Phosphodiester bonds run 5'->3' up one backbone strand in a double helix, and 5'->3' down the other.]
21.How many base pairs are there in one turn of the B form of DNA? [10.] What is the angular rotation (about the helical axis) between each base pair? [36.]
22.What form of DNA exists in solution in an isotonic buffer? [B form.] How was this determined? [By probing DNA absorbed onto calcium phosphate with DNase or hydroxyl radicals, or after circularizing linear DNA fragments of different lengths followed by measurement of the number of superhelical turns.]
23.What are the major and minor grooves in DNA? [Helical grooves running up/down the surface of the double helix.]
24.What is meant by melting (denaturing) DNA? [Separating the two strands in a double helix.] How can this be achieved? [Using high temperatures, acid or alkaline pH to destabilize H-bonds between complementary bases in the two strands, and stacking interactions between base-pairs.]
25.What is the melting temperature of DNA, and how can it be determined? [Melting in physiological buffers begins at ~55C in AT-rich regions, and progresses into GC-rich regions until complete at ~90C. The midpoint melting temperature (Tm) is ~70C. Melting can be monitored by following changes in optical absorbance or binding to hydroxyapatite.]
26.What is the rate-limiting step during reannealing of two complementary DNA strands? [The slow nucleation reaction.] What equation governs the rate of reannealing? [C/Co = 1/(1+ k.Cot), where C is the concentration of DNA that is single stranded at time t, C0 is the initial concentration, k is the reassociation rate constant.]
27.What properties of DNA are essential for its role as a carrier of genetic information? [DNA is chemically stable in solution, the four bases (A, C, G, and T) can be strung in any sequence along each strand in a triplet code, the specificity of base pairing ensures that each strand contains one copy of the information (allowing repair if one is damaged), individual strands can be separated, each strand can act as a template that can be copied into new a complementary molecule (allowing replication and transcription), and information can be exchanged between duplexes through recombination.]
28.Outline how genetic information can flow between DNA, RNA, and protein. [Cover: replication, transcription, translation, reverse transcription, repair, and recombination.]
29.How many base-pairs does a typical human chromosome contain? [~100 Mbp.] What is the contour length of such a DNA molecule? [~34 mm.]
30.What is the 'packing problem'? [The contour length of a DNA molecule that runs the length of the interphase chromosome must be considerably reduced to allow it to fit into a nucleus (~10,000-fold in a typical human cell).]
31.How was it shown that a single DNA molecule runs the length of a eukaryotic chromosome? [In yeast, using pulsed-field gel electrophoresis, and - indirectly - by sequencing the whole genome.]
32.How are DNA molecules of different sizes separated during gel electrophoresis? [DNA molecules carry a net negative charge and so move in an electric field through a matrix-free solution to the positively-charged electrode. However, differently-sized molecules travel at the same speed and are not resolved; while longer molecules contain more phosphates (and so charge) and can potentially move faster than shorter ones, they suffer more viscous drag and the two effects cancel each other. The gel matrix increases drag on longer molecules, slowing them enough to be resolved.]
33.What is pulsed-field electrophoresis? [Electrophoresis using an electric field that is reversed at specified intervals to increase separation of DNA molecules.]
34.Why are binding sites for proteins often phased every 10 bp along a DNA duplex? [The double helix makes one turn every 10 bp. As twisting the duplex is energetically unfavorable, binding sites are often found at multiples of 10 bp on one face of the duplex.]
35.How can a DNA duplex bend? [Most duplexes are either straight, or else they can bend in only one direction or plane (like a finger or a knee). However, some are very rigid (e.g., a run of (A)100 is so rigid it cannot be wrapped around a nucleosomal core), others more flexible (e.g., (TA)n). Still others are intrinsically bent (e.g., DNA with short runs of As spaced every 10 bp). At the more global level, DNA can be tied into loops and knots (e.g., it can make a smooth 90 turn over ~200 bp).]
36.What is DNA supercoiling? [Twisting of the axis of the duplex so that the molecule becomes torsionally strained; this supercoiling is in addition to the twisting of the two strands in the double helix.]
37.What does a DNA topoisomerase (type I and II) do? [Type I enzymes: cut one strand of the duplex, allow the cut end to rotate about the other (intact) strand, and reseal the cut. Type II enzymes: cut both strands in a duplex, pass an intact duplex through the gap, and rejoin the cut ends.]
38.Draw right- and left-handed toroidal and interwound supercoils. Include arrows indicating which forms can be interconverted without breaking covalent bonds.
39.How can energy in a supercoil be used to melt a DNA duplex? [The torsional energy spread throughout the superhelical molecule can be concentrated so that it can melt ~10 contiguous base-pairs.]
40.What equation describes the helical windings of a strand in a DNA molecule? [Tw + Wr = Lk, where Tw is the twist, Wr is the writhe, and Lk is the linking number.]
41.What is a 'restrained' supercoil? [The supercoiled state is a high-energy one, because torsional energy is lost spontaneously when even one phosphodiester bond anywhere in a DNA circle is broken; the cut end can rotate about the other strand in the duplex to release free energy of supercoiling. However, this torsional energy is 'restrained' (perhaps by binding to a protein complex) if it can no longer be released.] Provide an example. [E.g., the coiling of DNA in the nucleosome.]
42.How does the structure of RNA differ from that of DNA? [It contains U instead of T, and ribose instead of 2-deoxyribose. RNA often exists as a single strand (whereas DNA is generally double stranded), and it is usually more flexible (it can fold back on itself over a nucleotide or two).]
43.Draw the structure of a piece of RNA; highlight where the structure differs from that found in DNA.
44.Outline the path followed by the phosphodiester backbone in a typical tRNA molecule. Illustrate the positions occupied by the associated amino acid and mRNA.
45.Draw a base pair of DNA, and indicate which groups often interact with DNA-binding proteins.
Recognizing specific DNA sequences
46.Which assays are used to detect the specific binding of a protein to DNA? Describe how they work. [Filter-binding assay: relies on the affinity of DNA-protein complexes - but not free DNA - for nitrocellulose filters (DNA is usually labeled with 32P). Gel-shift assay: DNA sequence is labeled with 32P, mixed with a cell extract, the mixture subjected to electrophoresis through a polyacrylamide gel, and an autoradiograph prepared; DNA-protein complexes run more slowly than (smaller) free DNA through the gel (specific binding confirmed by competition with competitor oligonucleotides, and 'supershifting' using a specific antibody). DNA-affinity chromatography: proteins bind to the DNA on the column, and are usually progressively eluted by increasing the salt concentration. DNA footprinting: a pure DNA fragment containing the binding site is labeled at one end with 32P, treated with sufficient nuclease (e.g., DNase I) or chemical (e.g., hydroxyl radicals) to break each strand once, denatured, the resulting single-stranded fragments run on a sequencing gel, and those bearing the radiolabel are detected by autoradiography. A protein bound to a specific site protects underlying phosphodiester bonds from breakage so that labeled fragments that terminate in the binding site will be missing; this leaves a gap or 'footprint' in the sequencing ladder. Chromatin immunoprecipitation assay: cells are treated with formaldehyde to cross-link proteins to DNA, chromatin is isolated and sheared, and an antibody is used to immunoprecipitate the target protein and any associated DNA. After reversing cross-links, DNA is purified from the pellet, and specific DNA sequences are detected using the polymerase chain reaction.]
47.What is DNA 'footprinting'? [A pure DNA fragment containing the binding site is labeled at one end with 32P, treated with sufficient nuclease (e.g., DNase I) or chemical (e.g., hydroxyl radicals) to break each strand once, denatured, the resulting single-stranded fragments run on a sequencing gel, and those bearing the radiolabel are detected by autoradiography. A protein bound to a specific site protects underlying phosphodiester bonds from breakage so that labeled fragments that terminate in the binding site will be missing; this leaves a gap or 'footprint' in the sequencing ladder.]
48.Why are DNA-binding proteins often dimers? [The two strands in the double helix are antiparallel and rotationally symmetric. If a peptide can interact with one strand of the double helix, dimerizing that peptide in a rotationally symmetric way will ensure that the second peptide in the dimer will interact with the other strand. The fit will be perfect when a homodimer binds to a palindrome.]
49.Write out a palindromic DNA sequence. [ATGCGCAT]
50.Make notes on some DNA-binding motifs found in proteins. [E.g., SPKK motif, helix-turn-helix, zinc fingers, leucine zipper, helix-loop-helix.]
Making large structures
51.What are the general rules that govern the biosynthesis of all polymers? [Made from a limited number of different subunits. Subunits added one at a time in one direction, from a fixed starting point to a fixed terminus. One of two strategies used: activating group attached to the growing chain is released and replaced by the incoming unit (as in protein synthesis), or an activating group in the incoming unit is released (as in DNA and RNA synthesis).] What advantages does a strategy based on these rules provide? [Using one (or a few) identical subunits reduces the amount of information required to encode the structure. If subunits associate through multiple bonds of low energy, assembly and disassembly can be controlled more easily. Quality control over the final structure also becomes easier.]
52.Why are so many biopolymers helical? [Helices arise so frequently because assembly into a nonhelical structure requires that each subunit is positioned next to its neighbors with no axial rotation; even the slightest adds up over many subunits to give a helix.]
53.What is a molecular 'chaperone'? [Most biological structures assemble by themselves into larger structures; some (e.g., the icosahedral DNA bacteriophage P22) require help from an additional molecule that is not found in the final structure.]
54.Why do biologists use extracts of Xenopus eggs so frequently? [A mature oocyte (diameter of ~1 mm) contains all the precursors needed to make tens of thousands of cells and their nuclei. Therefore, it contains a highly-concentrated stockpile of many cellular precursors that can assemble functional nuclei and chromosomes.]
55.What is a 'minichromosome'? [A short piece of DNA - usually circular and ~5 kbp long - that is covered by regularly-spaced nucleosomes.] How can it be made in vitro, and what does it contain? [By adding naked, circular, DNA molecules to extracts of Xenopus eggs; minichromosomes contain DNA plus the four core histones (i.e., H2A, H2B, H3 and H4) assembled into nucleosomes.]
56.What properties of a protein ensure that it is localized to a particular part of a cell? [Nuclear, cytoplasmic, and extracellular proteins each have characteristic surfaces (cytoplasmic proteins have a balance of acidic and basic residues, extracellular proteins a slight excess of acidic residues, and nuclear proteins a pronounced excess of basic residues); then, we might imagine that newly-made proteins diffuse throughout the cell, to bind at a specific location. Individual proteins also have short peptide sequences (e.g., nuclear localization signals) that target the whole protein to a specific subcellular compartment (e.g., the nucleus).]
57.What is meant by 'tensegrity'? Give an example of a tensegrity structure, and what advantages do tensegrity structures have over more conventional ones? [Tensegrity: A design principle based on tensional integrity, rather than the compressional continuity required by (conventional) gravity-based architectures. Example: Geodesic domes are held up and open by interconnecting a continuous series of tension elements (e.g., elastic wires) with a discontinuous series of compression-resistant struts (e.g., steel beams). The structure persists independently of gravity, and individual elements need only to support one another locally.]
58.What determines the position of a nucleus in a cell? [Two major factors - the microtubules, and the microtubule organizing center. Where a nucleus is tightly associated with a microtubule organizing center (e.g., a male pronucleus) it inevitably follows the organizing center; where it is unattached (e.g., a female pronucleus) it can be pulled by molecular motors along microtubules.]
59.Why are nuclei often round? [Surface tension is a major determinant. The nucleus has a fluid system that is immiscible with its surroundings. Its surface tends towards the spherical minimum, and it is immersed in a medium that transmits on all sides a uniform fluid (hydrostatic) pressure; therefore, a nucleus is spherical.]
Some evolutionary considerations
60.Outline Darwin's theory of natural selection. [Any organism possessing a variation that enhances its chances of surviving is more likely than others to pass the variation onto its offspring. As only the fittest survive and reproduce, only their offspring go onto continue the struggle; but as they inherit the variation, they are more likely to succeed. The rest perish.]
61.What three generalizations underpin Darwin's theory of evolution by natural selection. [Individual members of any species vary one to another (both structurally and behaviorally), individual variation is to some degree hereditary, and organisms multiply at a rate that exceeds the capacity of the environment to carry them - with the inevitable consequence that many must die (the Malthusian principle).]
62.Make brief notes on pseudo-genes, junk DNA, retrotransposons, satellites, LINEs, and alu repeats. [Pseudo-genes: genes that are no longer functional. Junk DNA: non-coding DNA that is often assumed to be non-functional. Retrotransposons: short DNA sequences (1-10 kbp) that can move around the genome. Satellites: named because they floated as 'satellite' bands in a density gradient, rapidly-annealing tandem repeats, often found in heterochromatin, usually not transcribed. LINEs: constitute ~15% of human genome, encode a nucleic acid binding protein and endonuclease/reverse transcriptase, presumably able to transpose efficiently. Alu repeat: constitute ~10% human genome, contain ~300 nucleotides derived from 7SL gene encoding polymerase III promoter.]
63.What is a 'Cot curve'?[[Curve relating the fraction of single-stranded DNA that has renatured at time, t, to the initial concentration of DNA (Co) and time. It can be used detect repeated sequences, and to estimate roughly the amount of DNA in a genome.] How is the Cot1/2 related to genome complexity? [The higher the Cot1/2 the greater the genome complexity.]
64.Roughly, how much DNA is contained in the haploid human genome? [~3x109 bp.] How was this determined? [By measuring the amount of DNA purified from a known number of cells, quantitative micro-densitometry using Feulgen's procedure, and from the analysis of Cot curves.]
65.What is the size of a typical gene and exon in the human genome? [Genes have a mean size of ~20 kbp (median ~4 kbp), exons ~250 bp (median 135 bp).] How frequently are genes spaced along the chromosome? [Every ~60 kbp.]
66.Roughly, what is the genome size and gene number of E. coli, S. cerevisiae, C. elegans, D. melanogaster, and H. sapiens? [Genome size (Mbp): 4.6 (E. coli), 13.5 (S. cerevisiae), 97 (C. elegans), 180 (D. melanogaster), 3,500 (H. sapiens). Gene number: 4,200 (E. coli), 6,200 (S. cerevisiae), 18,400 (C. elegans), 13,600 (D. melanogaster), 70,000 (H. sapiens).]
67.What evidence indicates that most genes in the fly are expressed in many different tissues? [About 600 genes expressed in the larval brain were selected at random, and in situ hybridization showed that only two were expressed exclusively in the nervous system; most were expressed elsewhere, with little tissue specificity.]
68.Briefly describe the extra-nuclear DNA found in mammalian cells. [Each of the 100-1000 mitochondria in the cytoplasm of a human cell contains one or more copies of a circular DNA genome (16,569 bp) that encodes 2 rRNAs, 22 tRNAs and 13 different polypeptides.]
69.Draw diagrams illustrating the rate constants governing the interactions of two, three, four, and five interacting components. In each case, how many rate constants are involved? [2, 6, 12, 20.]
70.Describe the yeast 'two-hybrid' system and its uses. [Cover: the modular nature of the yeast GAL4 protein (with DNA-binding and activator domains), and transcription of a gene encoding a selectable marker and regulated by UASG.]
71.What is FRET, and how is it used? [Fluorescence resonance energy transfer (FRET): an interaction between excited states of two fluorochromes lying ~5 nm apart in the appropriate orientation so that energy is transferred from one to the other without emission of a photon. Use: Detection of the molecular 'touching' of two fluorescent probes (e.g., calmodulin tagged with the cyan variant of green fluorescent protein and a Ca2+-binding-peptide tagged with the yellow variant).]
72.What is meant by 'epistasis', and how is it used? [Epistasis: a one-sided interaction between two (non-allelic) genes (e.g., the expression of one gene masks the expression of another). Used: to detect interactions between the products of different genes.]
73.How does a bacterium swim toward a source of food? [Cover: periods of smooth swimming interrupted by 'tumbling', switching the direction of flagellar rotation, and biasing a three-dimensional random walk.]
74.Make short notes on why you think nuclei evolved. [Cover: the autogenous and exogenous pathways, and evolution from a sporulating bacterium.]
75.What is 'endosymbiosis'? [One organism lives within another organism, to the mutual benefit of both.]
Overview of nuclear structure
1.Write brief notes on the nucleolus, the nuclear envelope and pore, eu- and hetero-chromatin, facultative heterochromatin, and the Barr body. [Nucleolus: most conspicuous nuclear organelle, site where rRNA is synthesized, ribosomes assembled. Envelope: defining feature of eukaryotes, double membrane surrounding nucleus. Pore: 'gate' in nuclear envelope that allows communication between nucleus and cytoplasm. Eu- and hetero-chromatin: Different types of chromatin recognized by differential staining. Euchromatin is dispersed and occupies most nuclear volume. Heterochromatin is densely packed with fibers like those found in mitotic chromosomes, often condensed against nuclear membrane or nucleolus, but can aggregate into densely-staining, often internal, chromocenters. Facultative heterochromatin: expressed in some cell lineages but not others (e.g., X chromosome of eutherian mammals); distinguished from constitutive heterochromatin which is never expressed and often contains short DNA repeats. Barr body: dense body just under nuclear membrane containing inactive X chromosome.]
2.What are the stages of mitosis? [Prophase, metaphase, anaphase, telophase.]
3.How are 'spreads' of metaphase chromosomes prepared? [Collect blood in anticoagulant (e.g., heparin); stimulate to grow with phytohemaglutinin (lectin from red kidney bean); after 3 d add plant alkaloid, colchicine, for 2 h to arrest cells in mitosis; swell cells in hypotonic solution; treat with a mixture of glacial acetic acid and methanol to extract and fix; spread chromosomes by dropping onto a clean glass slide; stain.]
The nuclear membrane
4.Illustrate the relationship between the membranes of the endoplasmic reticulum and the nuclear envelope.
5.What is the nuclear lamina? [Responsible for determining nuclear shape, underlies nucleoplasmic side of inner membrane, made of a fibrous mesh of lamin proteins that are members of intermediate filament family.]
6.Describe the general structure of intermediate filaments (IFs). [Defining feature: central rod domain of four -helical regions (termed 1A, 1B, 2A and 2B) separated by three -helical linkers (L1, L12, L2). According to the degree of sequence homology, IFs grouped into six different categories; five represent cytoplasmic proteins expressed in a tissue- and differentiation-dependent manner, while the sixth includes nuclear lamins.] How do the lamins differ from the other IFs? [Possess a globular head domain and a tail domain with a nuclear localization sequence and a CaaX box. May be isoprenylated by addition of a farnesyl residue to the cys, subsequent cleavage of the three terminal amino acids, and isoprenylation. The cys may be further modified by carboxymethylation.]
7.Draw a cartoon illustrating the structure of the nuclear pore.
8.How was it shown that proteins of <9 nm can diffuse through the pore? [When fluorescent water-soluble molecules of ~5 kD microinjected into the cytoplasm, they quickly diffuse into the nucleus. Fluoresceinated larger proteins with diameters of <9 nm (e.g., ovalbumin of 43 kD) enter less quickly, while those of >9 nm (e.g., bovine serum albumin of 66 kD) cannot enter.]
9.Outline how proteins of >60 kD enter the nucleus. [Cover: the membrane as an impermeable barrier; entry through pore in an energy-dependent, saturable, selective process; roles of NLS, receptors, importins, Ran, RCC1; origin of directionality; illustrate with examples of SV40 T antigen, BSA tagged with fluorescein and NLS, nucleoplasmin.]
10.What are nuclear localization signals (NLSs), nuclear export signals (NESs), and nuclear retention signals (NRSs)? [Short peptide sequences in proteins that facilitate import (NLS) and export (NES) through the nuclear pore, or retention in the nucleus (NRS).] How are such signals identified? [NLS: cite mutational studies with SV40 T antigen, microinjection studies with BSA tagged with fluorescein and NLS, and with nucleoplasmin. Extend to NESs and NRSs.]
11.Describe how autoradiography can be used to identify which cells in a population are in S phase. [Grow cells on coverslips in [3H]thymidine so the analogue is incorporated into DNA. After fixation, unincorporated [3H]TTP is removed with trichloroacetic acid, the coverslips overlaid with photographic emulsion, left in the dark while the isotope decays, and the emulsion developed. Finally, cells are stained (through the emulsion) and viewed down a light microscope (again, through the emulsion). Silver grains can be seen in the emulsion, over nuclei that were in S phase.]
12.How is RNA exported through the pore? Provide experimental details of how the different mechanisms were uncovered. [Cover: microinjection of [32P]RNA into nuclei of Xenopus oocytes, analysis of the Rev response element (RRE) of HIV, and the role of the M9 sequence in HnRNPA1.]
13.Is the nuclear membrane an ion barrier? [Cover: the idea that membranes are usually impermeable to ions, the difficulties of measuring free ion levels in nuclei, and the presence of the machinery for regulating Ca2+ levels in nuclei.]
The nucleolus
14.What are the different types of ribosomal RNA, and which polymerases are responsible for their synthesis? [28S, 18S, 5.8S, and 5S rRNA. 28, 18, and 5.8S rRNA are made by RNA polymerase I, while 5S rRNA is made by RNA polymerase III.]
15.What are the main subcompartments within nucleoli, and what is their role? [Fibrillar center (store with active polymerases on the surface), dense fibrillar component (nascent transcripts), granular component (maturation site).]
16.Describe the organization of rDNA sequences in the human genome. [rDNA genes are arranged in tandem arrays. In man, arrays of ~40 rRNA genes (each encoding a 28, 18, and 5.8S rRNA) are found at the tips of chromosomes 13, 14, 15, 21, and 22, while ~2,000 genes encoding 5S rRNA are found on chromosome 1.]
Packaging chromatin during interphase
17.What is the 'packing problem', and how is it solved? [Problem: how to pack a long DNA duplex into a smaller nucleus (e.g., the contour length of a typical human chromosome is ~34 mm, nuclear diameter ~10 μm). Solution: compaction is achieved through a hierarchy of coiling (nucleosomes, zigzagging string, looping, 'clouds' of loops, chromosome territories).]
18.Why is it difficult to analyze nuclei and chromatin in isotonic buffers? [Interrelated problems: fragility of nuclei and the length of DNA (breaking even a few nuclei in a population releases long DNA strands that become entangled), high concentration and charge of nuclear material (facilitates aggregation), chromatin is poised in a metastable state (so small alterations in charge change compaction/aggregation).]
19.Describe how nuclei, 'matrices', 'scaffolds', and 'nucleoids' are usually isolated. [Nuclei: swell cells in hypotonic buffer, break cell membranes, pellet released nuclei free of cytoplasmic debris and membranes, wash nuclei by pelleting. Matrices: treat nuclei with 2 M NaCl to strip histones from DNA, detach most DNA with DNase. Scaffolds: Stabilize nuclei by thermal shock, extract most proteins with the strong detergent, lithium diiodosalicylate. Nucleoids: Lyse cells directly in a detergent (e.g., Triton X-100) and 1-2 M NaCl.]
20.Describe how 'matrix attachment regions' and 'scaffold attachment regions' are identified. [Prepare matrices or scaffolds, exhaustively treat with DNase I or restriction enzymes to remove most DNA, probe (by 'Southern blotting') to see which sequences in and around a particular locus of interest are present at a higher concentration in the remaining DNA (compared to total cellular DNA). Alternatively, clone and sequence the residual DNA fragments. MARs can also be identified by end-labeling DNA fragments from a locus of interest, and incubating them with matrices; MAR-containing fragments bind preferentially.]
21.What are the 'nucleosomal' 'core' histones? [H2A, H2B, H3, and H4.]
22.Outline the general structure of the nucleosome. What is the evidence for this structure? [Structure: 146 bp of duplex DNA wrapped around a histone octamer (two copies of H3, H4, H2A, H2B) in 1.65 turns of a flat, left-handed superhelix. Evidence: 2 copies of each core histone (and ~1 of H1) per ~200 bp DNA, the 'beads-on-a-string' structure seen by electron microscopy, nuclease digestion of linker DNA to give a nucleosomal ladder (repeat 180-260 bp) following by trimming of the linker to give the core particle with 146 bp DNA, the structure of the core particle determined by X-ray crystallography.]
23.How can the lengths of DNA in the nucleosome, and between nucleosomes, be determined? [By progressive digestion of chromatin with nucleases (e.g., micrococcal nuclease), purification of DNA, and sizing the resulting fragments by gel electrophoresis. The distance between 'rungs' in the resulting nucleosomal 'ladder' gives the repeat length, while the length of the most resistant fragments gives the length of DNA in the nucleosome. The length of the linker can be obtained by subtraction.]
24.Describe the evidence for and against the existence in vivo of a 'solenoid'. [For: electron microscopy of chromatin fibres in buffers of low ionic strength reveals helical solenoids with six to eight nucleosomes/turn and an 11 nm pitch. Against: solenoids are not seen at a physiological salt concentration, or in electron micrographs of rapidly-frozen whole cells.]
25.What is the evidence that the chromatin fiber is organized into loops in the interphase nucleus? [Cover: (i) it seems inconceivable that long DNA molecules are packed randomly like spaghetti, (ii) direct observation of lampbrush chromosomes (loops visible in unfixed material, but they might form when nuclei are dispersed), (iii) the demonstration of supercoiling in 'nucleoids' (but loops might be created artifactually during preparation), (iv) the rate at which nucleases release chromatin from interphase nuclei (but isolated in hypotonic buffers) or permeabilized cells (isolated in isotonic buffers). Go onto discuss the analysis of residual fragments, and how the results suggest that attachments (and so loops) change continually.]
26.Outline the path of DNA duplexes in the 'lampbrush' chromosomes of amphibia.
27.Describe how the contour length of a chromatin loop can be measured. [Permeabilize cells (ideally in an isotonic buffer, so describe the use of beads), treat with endonucleases, remove detached fragments, purify residual DNA, size on a gel, and calculate contour length.]
28.Describe how you would determine which sequences attach the chromatin fiber to the underlying structure in the nucleus. What are these attachment sequences? How stable are they? [Residual sequences are isolated as described above, and cloned and sequenced. Examination of the sequences shows that they are generally transcribed regions of the genome, or promoters or enhancers. Analogous experiments using minichromosomes confirm that transcription units are generally attached.]
29.What is an 'insulator', and how were they discovered? [Genes are often not expressed when inserted into the genomes of higher eukaryotes; they are 'silenced' by flanking genomic sequences. Insulators are able to prevent such silencing. They are discovered by inserting test sequences next to a reporter gene (e.g., white) to see if they increased its expression.]
30.What is 'position effect variegation', and how was it discovered? [The expression of a gene can depend on its position on a chromosome (e.g., proximity to other genes and heterochromatin). Therefore, a gene might always be active in one chromosomal context, but always inactive in another; its activity depends on its position. Occasionally, however, a gene is active in one cell, but inactive in another cell (even though the gene is in the same position, and the two cells are of the same type). This can lead to 'variegated' expression in cells of the same tissue. Such variegated position effects were discovered by Muller in 1930. He irradiated flies with X-rays, and found mutants with mottled red and white eyes. This phenotype was subsequently traced to the translocation of white from euchromatin to centromeric heterochromatin. Early during development, the primordia of the eye disc contain few cells. Inactivation of the gene in some cells - but not others - results in a mosaic pattern of expression. The activity (or inactivity) of the gene is maintained as cells divide; then, the mature eye - which contains ~800 regularly-arranged ommatidia - may contain patches with and without the normal red pigment.]
31.How is the 'territory' occupied by a chromosome during interphase identified? [Using in situ hybridization and chromosome-specific probes - a technique known as chromosome painting.]
32.What is the 'Rabl' orientation? [During mitosis, centromeres are pulled towards the two poles, and the nucleus initially forms around chromosomes that have centromeres at one end and telomeres at the other - the 'Rabl' orientation.]
33.What major factors determine the location of a chromosome within the interphase nucleus? [A 'memory' of chromosomal position from the previous mitosis when the chromosomes had the 'Rabl' orientation, the general 'stickiness' of heterochromatin and its tendency to aggregate at the nuclear membrane and around nucleoli, and the transcriptional activity of certain repeated genes - especially ribosomal genes - which leads to association with transcription factories; final position determined by resolution of these conflicting forces.]
34.What is FISH, and how would you use it to identify the location of a globin gene on a mitotic chromosome, and within an interphase nucleus? [Fluorescence in situ hybridization (FISH) is a technique that allows DNA sequences to be located relative to other landmarks; it can be applied to tissues, nuclei or chromosomes. In this case, spreads of mitotic chromosomes (or interphase cells growing on a coverslip) are fixed, and their DNA heated to denature it. Then the slide is incubated with a tagged DNA or RNA probe to allow it to hybridize with a complementary sequence. After removing excess probe, bound probe can be detected using the appropriate technique. For example, a probe tagged with biotin might be detected using an antibiotin antibody, and a secondary - fluorescently-labeled - antibody directed against the first. Sites where the probe has bound can then be seen in a fluorescence microscope.]
Nucleoskeletons and nuclear sub-compartments
35.Why has it been so difficult to observe the nucleoskeleton, and what are its constituents? [The density of chromatin in the nucleus obscures the underlying skeleton. However, underlying 'core' filaments with an axial repeat of 23 nm (typical of the intermediate-filament family) can be seen when most chromatin is removed; these filaments probably contain lamins. Nuclei may also contain other skeletal elements like actin.]
36.Make brief notes on nuclear bodies and dots, coiled (or Cajal) bodies, PML bodies, and PIKA domains. What are their roles? [Nuclear bodies: 0.3-1.5 μm in diameter, often found in nuclei of hyperstimulated or malignant cells. Coiled ('Cajal') bodies: subset of nuclear bodies, first identified by Cajal in 1903 after silver staining as nucleolar 'accessory bodies' (diameter 0.5-1 μm), contain coiled fibrils and autoantigen p80/coilin. Nuclear dots: 0.2-0.3 μm diameter, throughout nucleoplasm, often paired. PML bodies: originally detected using autoimmune sera from patients with primary biliary cirrhosis that labeled 5-15 intranuclear dots, contain PML protein. PIKA (polymorphic interphase karyosomal association) domain: diameter 0.5-5 μm, also recognized using autoimmune antibodies. Roles of all unknown.]
Chromosomes
37.What is an autosome, chromatid, and karyotype? [Autosome: chromosome other than sex chromosome. Chromatid: one of two sister chromatids that form a metaphase chromosome, contains a single DNA duplex that runs from one end to the other, sometimes used to describe the single unreplicated DNA duplex found in a diploid cell prior to S phase. Karyotype: complete set of chromosomes of a cell or organism, often applied to mitotic chromosomes arranged in homologous pairs.]
38.Outline the essential structural features shared by all eukaryotic chromosomes. [Each chromosome must contain a centromere, a telomere at each end, and one or more origins of replication.]
39.What is an 'autonomously-replicating sequence' (ARS)? How was the first one identified in yeast? [ARS: DNA sequence that enables a circular plasmid lacking an origin to replicate in yeast cells, usually equivalent to origin of replication. Discovery: Yeast mutants lacking the LEU gene cannot form colonies without added leucine, and even on transformation with a bacterial plasmid carrying the yeast LEU+ gene, few colonies result; this is because the plasmid is unable to replicate along with the yeast chromosomes and is soon diluted out. However, if random pieces of yeast DNA are inserted into the plasmid, a few will now contain a yeast replication origin and so can replicate in yeast cells. Now cells carrying such a plasmid will grow into a colony since they contain both the LEU+ gene and a yeast origin that facilitates replication of the plasmid.]
40.How would you show that a DNA duplex (~1 kbp) contained a region that could function as a centromere (or telomere) in yeast? [Centromere: stabilizes a LEU+, ARS+ plasmid in a leu- yeast cell, and enables it to persist despite growth in leucine. Telomere: enables a linear LEU+, ARS+, CEN+ plasmid to persist in a leu- yeast cell much like a normal yeast chromosome.]
41.What is a YAC, and how is it used? [YAC: yeast artificial chromosome. Use: as vector for cloning fragments of 0.1-1.5 Mbp DNA, usually in yeast.]
42.What are chromosome bands, and how are they produced? [Bands: After particular treatments, some reagents stain specific chromosomal regions - or bands - more intensely than others. These banding patterns are specific for individual chromosomes and serve as landmarks along the length of the chromosome so that chromosomes of similar size and shape can be distinguished. Q bands: detected using quinacrine, a fluorescent intercalator. G bands: seen after a brief heat treatment and staining with Geimsa. R bands: found after treatment with hot alkali before Geimsa staining.
43.Outline the 'scaffold' model for chromosome structure, and what is the evidence for it? [Model: An axial 'scaffold' containing topoisomerase II forms an underlying structure to which chromatin fibres are attached in loops. Evidence: Images of 'scaffolds' isolated from nuclei provide the best evidence for this kind of model; DNA loops are seen attached to the central axis.]
44.Outline the path of DNA duplexes in a 'polytene' chromosome.
3: REPLICATION
Principles
1.Draw the structures of the substrates and products obtained when one dNTP is added to a growing DNA chain by DNA polymerase.
2.Outline the principles involved in eukaryotic DNA synthesis. [Cover: restriction to S phase, semi-conservative replication, initiation at internal origins, simultaneous replication of many chromosomal segments as they move through polymerization sites in factories, strand separation to give a replication bubble flanked by two replication forks, requirements for primers and a primase, growth 5'-to-3', continuous and discontinuous synthesis on leading and lagging strands.]
3.Draw the structures seen by electron microscopy when T antigen is incubated with ATP and a linear duplex of DNA containing an origin.
4.How would you demonstrate that active DNA polymerases are fixed to an underlying structure in the nucleus? [Permeabilize cells, treat -/+ nuclease, remove detached chromatin, measure remaining polymerizing activity by incorporation of radiolabeled dTTP; removing most chromatin leaves most activity.]
Replication factories
5.Outline the different approaches used to label sites of DNA synthesis in eukaryotic nuclei, and the difficulties associated with each one. [By immunolabeling polymerases: not all enzyme active. By autoradiography with [3H]thymidine: slow entry and conversion to immediate precursor, dilution by endogenous pools, complications of rapidity of DNA synthesis, long path-length of -particles. By immunolabeling after incubation with Br-dU: slow entry and conversion, but higher resolution afforded by immuno-EM. By immunolabelling after permeabilization with immediate precursors like Br-dUTP and biotin-dUTP: control of elongation rate, but lysis might alter structure, and still limited resolution (even with immunogold labeling). Details of factories best seen after removing most chromatin.]
6.How were replication factories imaged in B. subtilis? [Using a PolC-GFP construct - one discrete spot is generally seen in the middle of the cell.]
The mechanics of synthesis at the fork
7.What is the unwinding problem, and how might it be solved in theory and in practice? [Problem: each strand in a DNA duplex is entwined about its partner, and must be untwined during replication. Theoretical solutions: by rotation about ends (but it these are fixed the two strands remain interlocked), by cutting one or other of the strands (or both), passing one (or both) strands through the break, and resealing the break. Practical solution: topoisomerases cut, pass, and reseal.]
8.Outline the reactions performed by type I and II topoisomerases. [Type I: transiently cleave single DNA strand in duplex to allow one (or both) cut ends to rotate around the intact strand, torsional tension drives rotation. Type II: transiently break both strands of a helix, structure of the yeast enzyme reveals dimeric protein with a large central hole - suggests model in which two sets of articulated jaws clamp two duplexes and pass one through the other, ATP drives reaction.]
9.Outline the role of DNA helicases in DNA synthesis. [A DNA helicase melts the two strands of the double helix to provide the single-stranded region that can be used by a polymerase. Uses ATP, unwinds in either 5'->3' or 3'->5' direction, but not both (T antigen is a 3'->5' helicase).]
10.Outline the role of single-strand binding (SSB) proteins in DNA synthesis. [Once the two strands have been separated, they are prevented from base-pairing again by SSB proteins.]
11.Outline the role of RNA primers in DNA synthesis. [DNA polymerases cannot initiate synthesis of new chains; they can only extend preexisting ones. Therefore, a special RNA polymerase - the primase - is used to make a short RNA chain (the primer) of 10-20 nucleotides, so a DNA polymerase can take over to catalyze addition of a deoxynucleotide onto the 3' OH of the primer.]
12.Draw template and nascent strands at a replication fork; label the 5' and 3' ends, and the leading and lagging strands.
13.What are Okazaki fragments, and how were they discovered? [Okazaki fragments: short strands of newly-made DNA copied during discontinuous (lagging-strand) DNA synthesis; they are ligated together shortly after synthesis. Discovery: Using T4 bacteriophage; infected bacteria were incubated at 8C for 5-50 sec in [3H]thymidine, DNA purified, denatured, and nascent chains sized by sedimentation in sucrose gradients. Initially, half the labeled chains were too long to be resolved, but the other half were 1,000-2,000 nucleotides. As the incubation time increased, the proportion of these short chains declined. These results are consistent with continuous synthesis of one strand and synthesis of the other in short pieces, followed by ligation.]
14.Draw a diagram illustrating the history (i.e., whether made by leading- or lagging-strand synthesis, or by replacement of an RNA primer) of the different pieces of DNA in a newly-replicated loop. Mark the 5' and 3'ends.
15.List as many different enzymic activities required during DNA synthesis as you can. [E.g., topoisomerases, helicases, single-strand DNA-binding proteins, clamp-loaders, circular clamps, primases, leading- and lagging-strand polymerases, RNase H activities, polymerases that fill in, and DNA ligases.]
16.What is a proofreading activity? [A 3'->5' exonuclease (either part of the catalytic subunit of a DNA polymerase, or a subunit of the polymerizing complex) that removes mispaired bases immediately after they have been incorporated.]
17.How is histone synthesis coupled to DNA synthesis? [Histones are synthesized mainly during S phase. This increased protein synthesis results from ~30x increase in mRNA levels due to a three- to five-fold increase in transcription, and a five-fold reduction in mRNA degradation. Once histones are made, they are probably assembled first into (H3-H4)2 tetramers; then H2A-H2B dimers are added, before the octamer binds to the nascent DNA. CAF-1 and Nap-1 facilitate assembly into nucleosomes.]
The initiation of synthesis
18.Describe the structure of the origin of replication in E. coli. [OriC contains: four 9-mers containing a specific recognition sequence (i.e., 5'-TTAT(C/A)CA(C/A)) for the initiator protein dnaA, three 13-mers that melt easily, 11 potential sites (i.e., GATC) of methylation by the Dam methylase, and 2 back-to-back promoters that may be involved in the initiation of replication.]
19.What information does DNA fiber autoradiography yield? [Bidirectionality of DNA replication from discrete internal origins, origin spacing, rate of fork movement, tendency of adjacent origins to fire together.]
20.How many origins does a typical human chromosome of 100 Mbp contain? [About 2,000 origins distributed every ~50 kbp.]
21.Outline the structure of the origin of SV40 virus. [This origin contains: four copies of the pentamer GAGGC organized as an inverted repeat (the prime binding site for the dodecameric T antigen), 17 AT base pairs likely to be the site where the duplex melts, an imperfect 15 bp palindrome, and signals for the initiation of transcription of 'early' and 'late' viral genes.]
22.What is an 'autonomously-replicating sequence' (ARS)? How was the first one identified in yeast? [ARS: DNA sequence that enables circular plasmid lacking origin to replicate in yeast cells, usually equivalent to an origin of replication. Discovery: The first ARS was obtained as follows. Yeast mutants lacking the LEU gene cannot form colonies without added leucine. Even on transformation with a bacterial plasmid carrying the yeast LEU+ gene, few colonies result; this is because the plasmid is unable to replicate along with the yeast chromosomes and is soon diluted out. However, if random pieces of yeast DNA are inserted into the plasmid, a few will now contain a yeast replication origin and so can replicate in yeast cells. Cells carrying such a plasmid will grow into a colony since they contain both the LEU+ gene and a yeast origin that facilitates plasmid replication.]
23.Outline the structure of a typical yeast origin. [Yeast origins often contain: an essential 11-nucleotide 'core consensus sequence' (the A element), plus three additional elements (B1, B2 and B3) that enhance ARS function. ORC binds to the A and B1 elements, while B3 is a binding site for ABF1.]
24.Outline two methods for mapping origins. [(i) Using 2-D gel electrophoresis. DNA is cut with a restriction enzyme, and fragments first separated by virtue of mass, and then according to shape. Linear fragments of different sizes lie along a diagonal, but Y-shaped forms (resulting from replication forks) and fragments containing replication bubbles (from the origin) move aberrantly and are offset from the diagonal. Specific DNA sequences are localized after 'Southern blotting' and hybridization with a labeled probe; maps of an origin and its surroundings can be built using different probes along the chromosome. (ii) Using PCR. Cells are grown in Br-dU so that nascent chains can be purified by virtue of their different density on gradients containing caesium chloride; then the chains are fractionated according to length, and the different fractions amplified by PCR using different pairs of primers that flank different regions. Primer pairs hybridizing to a region near an origin will amplify sequences in all size classes of nascent DNA, whereas those further away will only work with longer chains.]
25.How would you show that the same origins are used in a mammalian cell during successive cell cycles? [Cells are synchronized at the beginning of S phase just after early-replicating origins have fired, pulse-labeled with Br-dU, the label is removed, and the cells resynchronized at the beginning of the next S phase, pulse-labeled with I-dU, DNA fibers prepared, and Br-DNA and I-DNA immunolabeled with different colors. If the same origins are used during successive S phases, regions labelled with one color will also be labeled with the second.]
26.When during S phase is the DNA in R and G bands replicated? [R bands: early. G bands: late.]
Replicating ends
27.Outline the problem associated with replicating the ends of a chromosome, and some solutions. [Problem: A polymerase can extend a leading strand to the very end, but removal of a primer at the 5' end of the lagging strand leaves a gap that cannot be filled, as no 3'OH is available. Solutions: use protein-nucleotide priming (adenovirus), form a hairpin, concatamer, or circle (e.g., in vaccinia, T7 and lambdoid viruses), maintain ends by recombination (e.g., T4 bacteriophage), use telomerase.]
28.Outline the properties of telomerase. [Cover: it is part protein and part RNA, protein part has homology with reverse transcriptases, RNA part contains 8-30 nucleotides of RNA containing 1.2-1.9 copies of the C-strand repeat that templates synthesis of telomeric DNA.]
Principles
1.Draw the structures of the substrates and products obtained when one dNTP is added to a growing RNA chain by RNA polymerase.
2.Outline the basic principles involved in eukaryotic RNA synthesis. [Cover: transcription between promoter (start) and termination (stop) signals, multi-subunit polymerases in factories, the basic steps of transcription, initiation of synthesis of new chains, synthesis 5'-to-3'.]
3.How would you determine which parts of the genome are transcribed? [Using [32P]RNA (made by 'running-on' in [32P]UTP) to probe a 'blot', 'Miller' spreads, 'S1 mapping', and RT-PCR.]
Tracking versus immobile RNA polymerases
4.How would you demonstrate that active RNA polymerases are fixed to an underlying structure in the nucleus? [Permeabilize cells, treat -/+ nuclease, remove detached chromatin, measure remaining polymerizing activity by incorporation of radiolabeled UTP; removing most chromatin leaves most activity.]
5.How would you localize sites of RNA synthesis in eukaryotic nuclei? [Permeabilize cells, extend nascent transcripts in Br-UTP (or biotin-CTP), indirectly immunolabel sites containing the incorporated analogue with fluors or gold particles; labels are concentrated in discrete 'factories'.]
6.Describe the properties of the bacterial RNA polymerase. [Cover: the core enzyme (initiates poorly), σ (helps the core initiate), the holoenzyme, TATA and -35 boxes, closed and open complexes, rho independent and dependent terminators.]
7.What are the untwining and supercoiling problems, and how are they resolved? [Untwining problem (and solution): a tracking polymerase is likely to generate a transcript that is entangled about the template (fix the polymerase and allow DNA to rotate). Supercoiling problem (and solution): transcription by both tracking and fixed polymerases generates twin domains of supercoiling (role of topoisomerase).]
8.How was it demonstrated that attached RNA polymerases can work? [(i) Using a hybrid protein containing a polymerizing domain (from T7 polymerase) connected by a cleavable linker to an immobilizing domain; rates of elongation of the bound protein (attached to a plastic bead) and the free protein (released by cleavage with a protease) were similar. (ii) Using the polymerase from E. coli absorbed onto a glass slide, a template that had a promoter at one end and a gold particle at the other, and microscopy to monitor the distance between the gold particle and polymerase; the rate of transcription was deduced from the rate at which the distance between the two decreased.]
9.How would you measure the pulling power of a polymerase? [Using a polymerase absorbed onto a glass slide, a template with a promoter at one and a polystyrene bead at the other, and an 'optical tweezer'.]
10.Roughly, what force acting on DNA can stall a polymerase, strip a nucleosome from the template, and break the duplex? [About 14, 2, and 480 pN, respectively.]
The three kinds of eukaryotic RNA polymerase
11.How can the activities of RNA polymerases I, II, and III, be distinguished? [Using -amanitin; polymerase I is unaffected, polymerase II is very sensitive, and polymerase III moderately so.]
12.Which classes of RNA do RNA polymerases I, II, and III make? [Polymerase I: 5.8, 18, and 28S rRNA. Polymerase II: hnRNA (transcripts of most genes). Polymerase III: small RNAs, including 5S rRNA, tRNAs.]
13.Draw the general structures of the transcription units transcribed by the three eukaryotic polymerases.
14.How is a 'Miller' spread prepared, and illustrate the appearance of a spread containing some ribosomal cistrons? [Preparation: isolate nuclei, disperse chromatin in a hypotonic solution, spin onto a grid. Structure: series of 'Christmas' trees.]
15.Distinguish between: the core enzyme of RNA polymerase II and the holoenzyme, general and specific transcription factors, initiation and elongation factors. [Core/holo enzyme: minimal complex of 12-15 subunits able to carry out transcription / a large complex of >50 subunits containing both core enzyme and additional factors. General/specific transcription factors: facilitate initiation by the polymerase on most/specific genes. Initiation/elongation factors: stimulate initiation/elongation.]
16.Why is it so difficult to estimate the number of active polymerases in the cell? [Only a fraction of enzyme is active (i.e., ~25% in a HeLa cell), nascent transcripts are difficult to purify (they are present at a lower concentration than completed messages) and they have variable lengths (some have just initiated, others are about to terminate), transcription occurs very quickly (a typical transcript of ~7,500 nucleotides is completed in ~5 min), internal pools of >0.1 mM UTP dilute added radiolabels like [3H]UTP.]
17.Roughly, how many polymerases are associated with a typical transcription unit? [Polymerase I unit: ~125. Polymerase II unit: ~1.]
18.What is a DNA 'microarray?' Give an example of how one might be used. [Microarray: contains many different types of DNA (e.g., cDNAs or oligonucleotides) attached in a known array on a 2-D surface or 'chip'. Use: Arrays containing representative DNA fragments from all the genes in yeast can be used to assess which transcripts are present in a given cell population. Fluorescently-tagged cDNA copies of all the messages are made, and hybridized with DNA in the array; scanning the array for fluorescence then reveals which messages (cDNAs) are present.]
Transcription factories
19.What are the main subcompartments within nucleoli, and what is their role? [Subcompartments (role): Fibrillar center (store with active polymerases on the surface), dense fibrillar component (nascent transcripts), granular component (maturation site).]
20.Draw a cartoon illustrating the relationship between the structure of an active ribosomal cistron seen in a 'Miller' spread, and the structure found in vivo.
21.How would you label nascent transcripts in transcription factories? [Incubate living cells with Br-U (or permeabilized cells with Br-UTP or biotin-CTP), and indirectly immunolabel the resulting Br-RNA or biotin-RNA with fluors or gold particles.]
22.What is 'transcriptional interference', and how was it discovered? [Transcriptional interference: phenomenon where transcription of one gene prevents transcription of an adjacent gene. Discovery: Cells were transfected with a retroviral vector encoding resistance to neomycin and azaguanine, and clones harboring a single copy of the vector selected. Expression of the 3' gene was suppressed when selection required expression of the 5' gene, and vice versa. In addition, hardly any cells grew in both neomycin and azaguanine.]
Processing and transport of polymerase II transcripts
23.What are the main modifications made to a polymerase II transcript after it has been made? [Capping, splicing, and addition of poly(A).]
24.Draw the structure of a typical cap found at the 5' end of a message.
25.How can caps be isolated? [Exhaustively treat mRNA with endonucleases that cleave 3' phosphates next to bases (e.g., RNase T2) to leave 5' -> 5' links intact; purify resulting dinucleotides (each carrying several phosphates) free of mononucleotides on a column (separate molecules carrying different numbers of phosphate groups).]
26.Which radiolabels would you use to monitor capping? [Using ATP or GTP carrying 32P in the α, β, or γ positions (in vitro) - label in the α and β positions is retained in the cap; alternatively, using [3H]methionine (in vivo).]
27.What is the role of the cap? [The cap binds the cap-binding complex, CBC, which tethers the nascent transcript to the factory, enhances 3' end formation, protects transcripts from degradation, facilitates export from the nucleus, and dissociates at the ribosome to be replaced by the translational regulator, eIF-4E.]
28.What sequence motifs in RNA trigger polyadenylation in mammals? [The cleavage signal (AAUAAA) located 10-30 nucleotides 5' to the cleavage site, a U-rich or GU-rich downstream element 10-30 nucleotides 3' to the cleavage site, sometimes a U-rich upstream enhancer (USE), often a CA at the actual site of cleavage.]
29.What factors are required to constitute polyadenylation in vitro? [A four-subunit cleavage-polyadenylation specificity factor (which binds to AAUAAA), a three-subunit cleavage stimulation factor (CstF) that targets the DSE, two additional cleavage factors (CFI and II), poly(A) polymerase, and poly(A) binding protein II (PABII).]
30.What is the role of the poly(A) tail? [The tail facilitates export from the nucleus, increases the stability of some messages in the cytoplasm, and provides a recognition signal for the ribosome.]
31.What is an intron and exon? [Intron: an intervening or non-coding sequence of DNA within a gene that is transcribed into hnRNA but is then removed by splicing. Exon: the RNA sequence in a primary transcript (or the encoding DNA sequence) that exits the nucleus as part of a mature mRNA.]
32.How were introns discovered? [Cover: 'R-loop mapping' and the use of electron microscopy to visualize the 'R-loop', and comparison of the restriction maps of genomic and cDNA containing the globin genes.]
33.Why do you think introns evolved? [Presence of an intron enables a single transcript to be spliced in different ways to produce different mRNAs (and so different proteins), also probably facilitates recombination and evolution of new proteins through 'exon shuffling'.]
34.Draw the structure of the nucleotide at the branch-point in the lariat.
35.How would you demonstrate that some splicing occurs co-transcriptionally? [Using'Miller' spreads (the length of some extra-nucleolar transcripts on a transcription unit falls sharply), or RT-PCR (to measure the appearance of spliced intermediates after induction of the expression of the human dystrophin gene).]
36.What is 'nonsense mediated decay' (NMD), and how was it discovered? [NMD: mRNA with a stop codon in the normal position is stable in both nucleus and cytoplasm, but moving the stop codon near the 5' end leads to the loss - or NMD - of the message. Discovery: place stop codons at different positions in test genes (e.g., TPI) and then monitor transcript levels; stop codons close to the 5' end destabilize the transcript in both the nucleus and cytoplasm.]
The need for repair of damage
1.List the natural agents that commonly cause damage in our DNA. [Water (deamination, depurination), oxygen (through the superoxide radical, hydrogen peroxide, and hydroxyl radical).]
2.List the lesions most commonly found in our DNA. [Lost base (through depurination, depyrimidination), altered base or nucleoside (through deamination) 8-hydroxyguanine (through oxidation), M1G and etheno-A or -C (via lipid peroxidation), incorrect base insertion/omission (errors in replication), cyclobutane dimers (sunlight), strand breaks (ionizing radiation).]
3.What makes it so difficult to study the repair of damage in DNA? [Damage is infrequent, few agents induce only one kind of damage, almost any type of damage can be repaired, and repair systems are redundant.]
Some experimental approaches
4.What methods have led to our understanding of repair mechanisms? [Analysis of mutants deficient in repair (e.g., the three epistasis groups of S. cerevisiae - the excision repair RAD3 group, the post-replication repair RAD6 group, and the recombination repair RAD52 group; the ERCC genes of CHO cells, human XP patients), assays for unschedulated DNA synthesis (using [3H]thymidine and autoradiography, or Br-dU and immunodetection of Br-DNA), analysis of repaired DNA on density gradients containing CsCl, and biochemical fractionation and the development of cell-free systems.]
5.Make notes on repair defects and human disease. [Cover: sensitivity to sunlight and xeroderma pigmentosum (XPA-G defective in nucleotide-excision repair; XPV and polymerase ), Cockayne's syndrome (CS-B equivalent to ERCC6), trichothiodystrophy (some have mutations in XPB or XPD), ataxia telangiectasia (ATM kinase), Bloom's syndrome (defective DNA ligase activity), Fanconi's anaemia (chromosomal aberrations, sensitivity to cross-linking agents like mitomycin C).]
Types of repair
6.What are the basic biochemical steps found in many repair pathways? [Recognition of damage, excision of the lesion, DNA synthesis, ligation.]
7.What are the three main repair pathways found in man, and what makes them distinctive? [Direct restoration (bonds that link the lesion to DNA are broken directly, without breaking the phosphodiester backbone), base-excision repair (base excision, followed by DNA synthesis to repair the backbone, and ligation), nucleotide excision repair (patch excision, followed by DNA synthesis to repair the backbone, and ligation).]
8.Give an example of the events occurring during 'direct repair'. [SAM might alkylate DNA accidentally to produce O6-methylguanine. O6-methylguanine-DNA methyltransferase (MGMT) is a 'suicide' enzyme that repairs alkylated DNA by transferring a methyl group from O6-methylguanine to a cysteine residue in the enzyme by an irreversible reaction, 'killing' the enzyme.]
9.Outline the events occurring during 'base excision repair'. [Nonbulky base adducts (e.g., produced by methylating agents) and oxidized, reduced, or fragmented bases (from ionizing radiation and oxidative damage) are repaired by a battery of glycosylases able to recognize different kinds of base damage (e.g., a TpG resulting from deamination of a 5-methyl-C in a CpG). Such a TpG might be repaired as follows. TDG glycosylase recognizes the abnormal structure, kinks the DNA, and flips out the abnormal residue to accommodate the altered base in a specific recognition pocket that mediates hydrolysis of the glycosylic bond. Once the T has been removed, the phosphodiester bond 5' to the resulting abasic site is cut by APE1 that recruits DNA polymerase β. This releases the abasic sugar, inserts a C, and LIG3-XRCC1 seals the nick. DNA can now be restored to its original state by a DNA methyltransferase acting to give MeC.]
10.Outline the events occurring during 'nucleotide excision repair'. [Bulky adducts (e.g., benzo[a]pyrene-guanine adducts caused by smoking, thymine dimers and (6-4) photoproducts induced by uv light) are removed in a series of reactions: recognition of the damage (by XPC), recruitment of TFIIH, XPA, and RPA (to open the helix, verify the lesion, and provide a landing pad for the rest of the machinery), coupled incisions (one on each side of a lesion to release an oligonucleotide carrying the damage) by an excinuclease (containing an ERCC1-XPF complex that cuts ~24 phosphodiester bonds 5' from the lesion and XPG that cuts 5 phosphodiester bonds 3'), DNA synthesis (probably by DNA polymerase δ or ε to fill in the gap, and ligation to restore strand integrity. One major pathway operates throughout the genome, another is coupled to transcription.]
11.How was the size of the 'patch' of DNA removed during nucleotide excision repair determined? [A DNA substrate was made by hybridizing an oligonucleotide containing a thymine dimer with a single-stranded circle of plasmid DNA, extending the oligonucleotide with DNA polymerase, and ligating to give a double-stranded circle. This substrate was incubated with a cell-free extract, modified dNTPs containing 5'-[α-thio]triphosphates, and a trace of [32P]dCTP; the excinuclease removes the dimer and flanking nucleotides, while DNA polymerase fills in the gap. Sulphur in the repaired phosphodiester backbone makes it resistant to exonuclease III but sensitive to iodine, while the radiolabel allows autoradiographic detection of repaired templates amongst the excess of unrepaired ones. The product is now cut with various restriction enzymes, treated ± exonuclease III or iodine, run on a gel, an autoradiograph prepared, and fragments sized. The limits of the repaired patch are mapped relative to the known positions of the oligonucleotide and restriction sites.]
12.Outline the events occurring during 'mismatch repair'. [T might be inserted opposite a G during replication, and mismatch repair corrects such mistakes. In bacteria, MutH, MutL, MutS, and MutU (in conjunction with helicase II, exonuclease I or VII, DNA polymerase III, and DNA ligase) recognize, remove, and then repair any mismatches in an unmethylated strand. Human cells contain homologs of these proteins, and some of these homologs (e.g., hMSH2, hMSH6, hMLH1, hPMS1, and hPMS2) are often mutated in colorectal cancer.]
13.What are the three major damage response systems in bacteria? [The SOS system: initiated by single-stranded DNA generated during repair, inactive RecA protease binds, promotes cleavage of LexA repressor and so transcription of genes involved in recombination (recA), repair (UvrA, UvrB) and replication (dnaQ, polB); as a result, cells stop dividing and repair lesions faster or remove them by recombination. The adaptive response to alkylating agents: bacteria adapt to grow in low concentrations of alkylating agents; Ada is activated by alkylation to turn on genes involved in inactivating the alkylating agent (aidB) or eliminating the lesion (ada, alkA). The adaptive response to oxidative stress: induced by reactive oxygen species by oxidation of OxyR (induces a conformational change so stimulating transcription of the catalase and alkylhydroperoxide reductase genes), or oxidation of the Fe4S4 center in SoxR by superoxides (induces transcription of the gene encoding SoxS, which stimulates transcription of genes encoding detoxifying enzymes like MnSOD and repair enzymes like endoIV AP endonuclease).]
Sites of repair
14.How would you determine whether the DNA polymerases involved in repairing damage induced by uv-irradiation were attached to the underlying structure in human nuclei? [Human cells would be uv-irradiated, allowed to initiate repair, and lysed. If polymerases diffused to lesions to carry out running repairs, [32P]dTTP should be incorporated into repaired patches wherever they were in chromatin loops. After cutting loops into small pieces, electrophoresis should remove any detached fragments (and any incorporated 32P they contained). However, if repairing polymerases were concentrated in factories, the radiolabel would not be detached and lost. Results were consistent with some repair taking place out in the loop, the rest in factories.]
15.Outline the evidence that some repair is coupled to transcription? [(i) In mammalian cells transcribed regions are preferentially repaired (pyrimidine dimers are removed five-fold more quickly from the transcribed DHFR gene of CHO cells than from bulk DNA; CS cells lack this kind of repair). (ii) In E. coli, the template strand is also repaired preferentially (TRCF recognizes and release a stalled complex containing RNA polymerase and the damage recognition subunit of the excinuclease). (iii) Many transcription factors turn out to be repair factors (e.g., microinjecting TFIIH into XPB and XPD cells corrects their deficiency in UDS). (iv) Some (but not all) repair sites are transcriptionally active.]
Some consequences of inefficient repair16.What are the main consequences of introducing damage into the DNA of mammalian cells? [Three pathways are triggered - DNA repair, cell cycle arrest, and apoptosis. If cells with damaged DNA continue to divide, mutations become fixed, and cancer may result.]
17.Outline the central features of the somatic mutation theory of cancer, and the evidence for it. [Features: carcinogens initiate neoplastic transformation by damaging DNA, some of the resulting lesions remain unrepaired to generate mutations in the descendants of the affected cells, some may occur in genes regulating growth, and this eventually leads to cancer. Evidence: epidemiology (exposure to specific environmental agents that damage DNA correlates with a high incidence of human cancers), many carcinogens are mutagens (screen for mutagens using the Ames test), many DNA damaging agents (ionizing radiations, uv light, various chemicals) transform mouse fibroblasts in tissue culture, so that they cause cancer when injected into mice.]
6: REGULATION OF GENE EXPRESSION
Simple regulatory circuits in bacteria and yeast
1.Differentiate between positive and negative control mechanisms in bacteria. [Positive: binding of an activator (e.g., cAMP) to a DNA-binding protein (e.g., CAP) stimulates that latter's DNA binding, and so initiation by RNA polymerase. Negative: binding of a repressor (e.g., the trp repressor) promotes DNA binding, preventing initiation.]
2.Outline the effects that occur when tryptophan switches off expression of the trp operon. [Two molecules of tryptophan bind to the trp repressor (a helix-turn-helix homodimer), increasing its affinity for the operator; the two recognition helices tilt and enter the major groove about 10 bp apart to contact the edge of the relevant bases. Now, the bound complex prevents the template from attaching to the polymerase. When tryptophan is absent, the now-unoccupied repressor dissociates from the operator so the template can attach productively to the polymerase (and the operon is transcribed).]
3.How does the catabolite activator protein promote expression of catabolic enzymes in bacteria? [Falling glucose levels increase the concentration of cAMP, which binds to CAP (a homodimer of a polypeptide containing a helix-turn-helix motif), promoting its affinity for target sequences embedded in the promoters of many genes encoding catabolic enzymes. Binding in the major groove narrows it, while the opposing minor groove widens; the result is a 40 kink. Now promoters attach more efficiently to polymerases, so catabolic enzymes are expressed at higher levels.]
4.Draw cartoons illustrating how the lac and lambda repressors regulate the activity of their respective operons.
5.How was it demonstrated that GAL4 has two distinct functional domains? [(i) If UASG is placed as far as ~600 bp upstream of another yeast gene, that gene can now be turned on by GAL4. (ii) When the C-terminus is deleted, DNase footprinting shows that the truncated protein still binds to UASG without activating transcription. (iii) Replacing the N-terminus with the bacterial repressor, LexA, ensures that yeast genes associated with a lexA operator are brought under the control of the modified GAL4 (showing the activation domain is functionally independent of the DNA-binding domain).]
Principles of eukaryotic gene regulation
6.How would you test the totipotency of the nucleus in a frog fibroblast? [(i) Uv irradiate an unfertilized frog egg to inactivate the resident nucleus. (ii) Microinject the nucleus from the fibroblast into the egg, and allow the egg to develop into a blastula. (iii) Remove the nucleus from one of the cells in the blastula, transfer into another uv-irradiated egg, and allow the egg to develop. As most eggs fail to develop into tadpoles or frogs, repeat. However, some will develop into tadpoles and frogs, showing that they received totipotent nuclei. Check that the nuclei in such tadpoles and frogs are genetically identical to those in donor fibroblasts, and that there were too few undifferentiated cells (lacking vimentin detectable by immunofluorescence) in the donor population to account for the observed success rate.]
7.How was 'Dolly' the lamb cloned? [Dolly's genes are derived from the udder of a (white-faced) Finn Dorset ewe. Cells from this ewe were arrested by serum starvation in G0, fused (using an electrical pulse) with an enucleate egg (from a Scottish Blackface ewe) in meiotic metaphase II, and the reconstituted egg cultured in the ligated oviduct of a (Scottish Blackface) foster mother for 6 days. Then, the egg was recovered, checked to see that it had developed into a blastocyst, reimplanted into a second (Scottish Blackface) foster mother, and allowed to develop to term (giving white-faced Dolly). Dolly's nuclear genes (excepting any mutant genes) are identical to those of her genetic mother (but not her foster mother.]
8.How were the rearrangements that occur in immunoglobulin genes during the development of B cells discovered? [DNA from a whole mouse embryo was cut with a restriction enzyme and 'Southern' blotted onto a filter; hybridization with the appropriate probes shows that V and C regions are on DNA fragments with different lengths. However, when the experiment is repeated with DNA from myeloma cells making a specific Ig light chain, the two regions are found together on the same fragment.]
9.Draw a cartoon illustrating the genetic changes that might occur during lymphocyte development in the gene encoding the mouse κ light chain.
10.By what mechanisms might selective gene expression be achieved? [E.g., DNA rearrangement (immunoglobulin genes) or amplification (rDNA genes) or loss (Dipteran embryos) or modification (methylation), alteration in the rate of transcriptional initiation (many genes) or elongation (heat-shock locus), transcript rearrangement (alternative splicing) or processing or degradation (histone mRNA), differential transport of mRNA out of the nucleus or to different locations (-actin), differential translation of mRNA, differential protein degradation or stabilization.]
11.List some examples of non-genic transcripts that control gene activity. [E.g., XIST RNA (maintains inactivity of X chromosome), LCR transcripts (α- and β-globin, keratin 18), let-7 transcripts (lin genes in the worm), transcripts involved in Ig switching.]
12.How do restriction enzymes get their name? [Restriction methylases and endonucleases recognize specific DNA sequences and constitute a system that defends bacteria against viral infection: the methylase attaches a methyl group to an A or C within (or close to) the recognition sequence, while the nuclease generally cuts the unmethylated sequence. Levels of methylase and nuclease produced are set at levels ensuring that the bacterial chromosome remains intact, while invading (unmethylated) viral DNA is cut. Therefore, viral growth is 'restricted' to strains lacking the nuclease. However, viruses can become infective if grown ('modified') before infection in a strain containing the methylase.]
13.Draw the changes that 'establishment' and 'maintenance' methylases might make in a CpG sequence before and after replication.
14.How would you demonstrate that globin genes in a myoblast (but not those in an erythroblast) were methylated? [By comparing the way HpaII (cleaves CCGG but not CMeCGG) and MspI (cleaves both CCGG and CMeCGG) cut DNA from the two sources. DNA is isolated, cut with one or other enzyme, fragments resolved by electrophoresis, 'Southern' blotted, and hybridized with a globin probe; the globin gene in erythrocyte DNA is cut by both enzymes (and so is unmethylated), whereas the myoblast gene is cut by MspI, but not by HpaII.]
15.Draw a cartoon illustrating the way alternative splicing of sxl transcripts determines sex in Drosophila.
16.What is RNA interference? [The suppression of gene expression by an RNA molecule.]
17.Draw a cartoon illustrating the differences in activity of the X chromosome in different cell lineages in a normal human female.
18.How would you demonstrate that different X chromosomes were active in cells from different parts of the head of a normal female (assuming she was heterozygous for the 'fast' and 'slow' variants of G6PD)? [Pull single hairs from different parts of the head, and determine the electrophoretic mobility of the G6PD in each root tip. Analysis of many tips would show that most contain only the A or B form, with very few expressing both.]
19.Give an example of an experiment showing that differential expression of a gene can require continuous regulation? [A typical muscle cell expresses the cell-adhesion molecule, N-CAM, on its surface. The stability of the switches involved in maintaining N-CAM expression were analyzed by fusing a mouse myoblast with a human lung fibroblast (which does not express any muscle-specific proteins); the resulting heterokaryon expressed N-CAM of both species. This suggests that a switch acting continuously on the muscle nucleus can also switch on N-CAM in the human nucleus.]
20.What is 'subtractive' hybridization' of a cDNA library? Outline how it was used to identify myoD. [Subtractive hybridization: subtraction of irrelevant cDNAs in a library to leave an enriched population. Identification: Fewer than 0.01% 10T1/2 cells differentiate spontaneously into myoblasts, but ~25% can be induced to do so by treatment with 5-azacytidine. A cDNA library made from the drug-treated cells (which contain considerable amounts of myoD cDNA) was radiolabelled, and hybridized with excess mRNA from untreated cells; myoD cDNA becomes enriched when duplexes were removed. The residual cDNAs were then used to probe for complementary clones in a myoblast cDNA library. Ninety-two cDNAs surviving this first screen were then used in the second screen. They were again radiolabeled, and hybridized with RNA from untreated and drug-treated 10T1/2 cells; myoD was one of three cDNAs that hybridized only to RNA from drug-treated cells. All three cDNAs were now transfected (with a linked selectable marker) into untreated 10T1/2 cells, and found to induce myosin expression. The myoD gene was recovered from one of the transfectants, and recloned; it promoted myogenic conversion at a high frequency.]
21.How was it shown that the activity of a eukaryotic gene can be regulated over eight orders of magnitude? [Rat growth hormone (rGH) can be detected with high sensitivity using a specific antibody; it is expressed in somatotrophes of the anterior pituitary, but not in hepatoma cells. The two kinds of cells were grown briefly in radiolabeled amino acids before rGH was immunoprecipitated, run on a 2-D gel and an autoradiograph prepared; the intensity of the resulting spot then reflected the amount of rGH made during the pulse. Reconstruction experiments showed that 4 molecules of rGH could be detected per cell. In somatotrophes, 108-fold more hormone was made than in hepatoma cells. No rGH message could be detected in hepatoma cells by hybridization with a radiolabeled probe; again reconstruction experiments showed that somatotrophes had 107 more rGH messages than liver cells. Assuming that mRNA is equally stable in the two cell types, then transcription rates can also be controlled over ~8 orders of magnitude.]
Regulation at the level of the nucleosome
22.List possible ways that the initiation of transcription might be regulated at the nucleosome. [(i) All nucleosomes are stripped from the template, so the promoter can bind to the polymerase. (ii) A nucleosome covers the promoter, preventing binding. (iii) Nucleosomes are 'positioned' so the promoter remains accessible. (iv) The structure of a nucleosome at the promoter is altered (e.g., through histone acetylation or phosphorylation).]
23.How would you distinguish between DNase 'sensitive' and 'hypersensitive' regions in chromatin? [Nuclei are incubated with DNase I, their DNA isolated and recut with a restriction enzyme, before resulting fragments are run on a gel. The amount of a particular gene that remains intact is then determined by 'Southern' blotting and hybridization with a radiolabeled probe; the intensity of the resulting band in the autoradiograph reflects the fraction of the gene that resists degradation by the nuclease. Most transcriptionally-genes are covered with nucleosomes, but more sensitive to digestion than inactive genes; they are DNase 'sensitive' and give weaker bands in the autoradiograph. Certain sites ('hypersensitive sites') within them are especially sensitive, and cutting within them releases fragments that are shorter than those released by cutting solely with the restriction enzyme.]
24.Where are histones acetylated? [At the N-terminal tails, on terminal serines or internal lysines.]
25.List the different covalent modifications of histones. [Acetylation, phosphorylation, methylation, poly(ADP)ribosylation, and ubiquitination.]
26.How are chromatin remodeling machines detected? [By an ATP-dependent change in accessibility of DNA in a nucleosomal array to nucleolytic digestion.]
27.List some chromatin remodeling machines. [E.g., the yeast SWI/SNF complex, ACF, NURF, CHRAC, histone acetylases and deacetylases like NuRD and RSF, RNA polymerase II.]
Regulation at the level of the loop
28.What is an enhancer and locus control region (LCR)? [Enhancer: A DNA motif or control element that acts over a distance of >1kbp to stimulate transcription of an associated gene when positioned in either orientation on either side (i.e., 5' or 3') of the gene. LCR: A DNA motif or control element similar to an enhancer that also acts over a distance, but which also has the additional property of overcoming position effects to insulate the transgene from the repressive effects of neighboring heterochromatin.]
29.How would you perform a 'run-on' transcription reaction, and for what is it used? ['Run-on' reaction: cells are permeabilized, and incubated briefly with radiolabeled nucleotide triphosphates so nascent transcripts are elongated by a few nucleotides. Use: measurement of the amount of transcription of a specific gene (after purifying labeled RNA, the concentration of nascent transcripts of the gene in question can be measured by hybridization with specific probes).]
Heterochromatin
30.List some properties of heterochromatin. [E.g., densely-staining, transcriptionally inactive, rich in repeated sequences, inaccessible to nucleases.]
31.What is meant by the term 'silencing' in yeast? [The transcriptional inactivation of a gene (e.g., the MAT locus) on insertion of the gene into certain regions of the genome.]
32.What is a 'CpG island'? [A region (often hypomethylated) of genomic DNA rich in the dinucleotide CG, and which is found upstream of a housekeeping gene.]
Establishing and inheriting patterns of expression
33.Illustrate how the activity of rDNA genes can be inherited through mitosis. [Cover: NORs, tandem repeats of 45S rRNA genes carried on five pairs of human chromosomes with perhaps only 6 loci being transcribed, activity associated with UBF on mitotic chromosomes, those NORs carrying UBF tend for form nucleoli in daughters.]
34.How would you demonstrate the effects of a maternal effect gene in Drosophila? [Cross the (grandparent) fly carrying the mutant maternal-effect allele with another fly carrying the same mutation (i.e., -/+ x -/+). Although one-quarter of the resulting fertilized eggs are genotypically -/-, they contain maternal products of the + gene, so the embryonic body plan is laid down normally. When such embryos reach adulthood they can be crossed with a wild-type male (i.e., -/- x +/+); then, the resulting fertilized eggs have +/- genes in a cytoplasm that lacks any + products from the mother. As a result, an apparently normal mother lays an egg that then develops abnormally.]
Overview
1.List the phases of the mammalian cell cycle. [G1 (and G0), S, G2, and M.]
2.How was the cell cycle discovered? [Grow bacteria for about one-tenth their generation time in a DNA precursor (e.g., [3H]thymine), and autoradiography reveals that most cells become labeled; in the equivalent experiment, only 20-30% mammalian cells become labelled (using [3H]thymidine) so most are not replicating.]
3.How would you obtain mammalian cells synchronized at different phases of the cell cycle? [In mitosis: by 'shake-off' after treatment with agents like colcemid, vinblastine, nocodazole, or nitrous oxide to block spindle operation. In G1: by virtue of small size using a fluorescence-activated cell sorter, or centrifugal elutriation (perhaps after isoleucine deprivation). In G0: by serum starvation. In S phase: using reversible inhibitors of DNA synthesis (e.g., high concentrations of thymidine, hydroxyurea, aphidicolin). In G2: using staurosporine.]
Mitosis
4.Define mitosis. [The process of nuclear division in the somatic cells of eukaryotes.]
5.What is cytokinesis? [The process by which the cytoplasm of a cell is divided once nuclear division (mitosis) is complete.]
6.What are the stages of mitosis? [Prophase, metaphase, anaphase, telophase.]
7.Illustrate the changes that occur to a chromosome as it progresses around the cell cycle.
8.Make notes on microtubules. [Cover: one of the three main filament systems of the cytoplasm that serve as highways for motor proteins (kinesin, dyneins), long hollow rods (25 nm diameter) made of tubulin (heterodimer of α and β subunits), each tubule has a growing (plus) and shrinking (minus) end, minus end usually stabilized by attachment to microtubule-organizing center (centrosome), role of GTP in binding to the β subunit and regulating length, effects of colchicine on binding to a tubulin monomer and preventing it from polymerizing to arrest cells in metaphase.]
9.What is the centrosome, or microtubule organizing center? [The centrosome surrounds the centriole in animal cells. It organizes the cytoskeleton, and so controls cell motility and shape. It also divides in two to organize the two poles of the mitotic spindle.]
10.What is the centriole, and what are its roles? [The centriole is an organelle in an animal cell made of up of two orthogonally arranged cylinders; nine groups of three microtubules form the wall of each cylinder, with each triplet tilted inwards like a turbine blade. An almost identical structure is found in the basal body of a cilium. Pericentriolar material is the major microtubule organizing center in the cell, so that the centriole indirectly organizes the cytoskeleton, and so influences cell motility and shape. Both the centriole and associated microtubule organizing center also divide into two to organize the two poles of the mitotic spindle; therefore, they also control chromosome segregation.]
11.Illustrate the centrosome cycle in an animal cell.
12.What is a kinetochore? [The kinetochore is a button-like structure found at the centromere of a chromosome; attachment of spindle microtubules connect it to the centrosome.]
13.List the different types of microtubules found in the spindle at metaphase. [Astral, kinetochore, polar.]
14.Draw a spindle with attached metaphase chromosomes; mark the different kinds of microtubules.
15.List the requirements for exact segregation of chromosomes. [The two kinetochores on sister chromatids must attach to microtubules extending from opposite poles (physical pairing between chromatids ensures that their kinetochores face in opposite directions), bi-oriented chromosomes must align midway between the two poles (to ensure that both partners reach target poles in time to be included in daughter nuclei), oppositely-oriented sister chromatids must separate and move to their respective poles at anaphase.]
16.List the roles of centromeres during mitosis. [The centromere is (i) the site of formation of the kinetochore and the point of spindle attachment (and so a structure that regulates attachment to the spindle), (ii) the last point of contact between sister chromatids at the metaphase-anaphase transition (and so it contains the machinery necessary to effect the final separation), (iii) a center of cell-cycle checkpoint control (and regulates chromosome separation), and (iv) a marshaling area for 'passenger' proteins that transfer from chromosomes to the spindle during metaphase or anaphase.]
17.How would you classify centromeres? [In some chromosomes (e.g., in C. elegans), centromeres (the attachment point of spindle microtubules) are scattered along the entire length of chromatids. In others, centromeres are localized to a single region of a chromosome arms; these can be classified into point (e.g., in S. cerevisiae) and regional centromeres (e.g., in S. pombe and man).]
18.Outline the structure of a centromere of S. cerevisiae. Which of its elements are essential? [A typical centromeric (CEN) locus consists of ~110 bp containing three DNA elements (CDEI, II, and III) that are sufficient to allow faithful segregation. CDEI and III (8 and 26 bp, respectively) are separated by CDEII, a poorly-conserved AT-rich 'spacer' (78-86 bp). CDEIII is absolutely required for centromere function; mutations in it disrupt chromosome segregation. Deletions of CDEI and CDEII only slightly affect mitotic centromere function, but sister chromatids sometimes separate precociously during the first meiotic division or mis-segregate during the second.]
19.Outline the structure of a human centromere. [Human centromeres are regional centromeres that extend over >1 Mbp. They contain thousands of copies of the -satellite repeat, which contains a binding site (17 bp) for CENP-B. During mitosis, the centromere organizes a kinetochore, which appears in the electron microscope as a three-layered disc; most centromeric chromatin (and CENP-B) are found underneath an 'inner plate,' separated from an 'outer plate.']
20.How did Nicklas and Koch (1960) show that the tension generated by conflicting forces guides chromosome pairs toward the correct bipolar alignment? [Cover: the use of chromosomes of grasshopper spermatocytes aligned on the metaphase plate during meiosis I, stable attachment of the two kinetochores of the duplicated chromosome to opposite poles, the loss of stability when two kinetochores attached to the same pole, stabilization by pulling on incorrectly-aligned chromosomes.]
21.What are the two classes of microtubule motor? [Dyneins, kinesins.]
22.Illustrate how minus- and plus-end-directed motors might (i) move a chromosome, (ii) 'focus' minus ends of two microtubules at a centrosome, (ii) maintain a constant length of a kinetochore tubule, and (iii) cross-link antiparallel polar tubules at the equator.
23.Outline the steps that occur during cytokinesis. [Cover: the formation of a contractile ring of actin and myosin, the generation of a cleavage furrow, and the formation of the mid-body.]
Regulation of the cell cycle
24.In which phases of the cell cycle are the major checkpoints in budding and fission yeast? [Budding yeast: G1 (START). Fission yeast: G2 checkpoint.]
25.What are the two major components of maturation promoting factor (and of the G2 checkpoint machinery in fission yeast)? [A cyclin-dependent protein kinase (CDK) that phosphorylates selected serines and threonines on proteins, and an activating cyclin that accumulates in a cycle-dependent manner.]
26.Outline how adrenaline regulates glycogen levels in skeletal muscle. [Cover: Glycogen is the major storage form of glucose, degradation regulated by adrenalin, 'fight or flee' response, binding to β-adrenergic receptors on cell surface stimulates rise in cytoplasmic cAMP, activates glycogen phosphorylase to break down glycogen to glucose-1-phosphate - resulting glucose provides energy source for muscle contraction. Cascade of A kinase, phosphorylase kinase, and glycogen phosphorylase. 'Catalytic' subunit of A kinase bound to 'regulatory' subunit in an inactive complex. When cAMP levels increase, binding to regulatory subunit induces conformational change that releases catalytic subunit that phosphorylates phosphorylase kinase, to activate it to phosphorylate key serines on glycogen phosphorylase, activating it in turn; this then degrades glycogen. 'A' kinase also phosphorylates (and so inhibits) glycogen synthase.]
27.Outline the various ways that the catalytic activity of the different members of the human 'kinase' family are regulated. [(i) Addition of extra controlling subunits or domains (e.g., cAMP-dependent protein kinase), (ii) addition of extra subunits whose level of expression varies depending on functional state of the cell (e.g., cyclin regulation of CDKs), (iii) addition of extra domains that target the kinase to different molecules or subcellular compartments (e.g., SH2 and SH3 domains of Src kinases), (iv) addition of extra domains that inhibit kinase activity by an autoregulatory process (e.g., myosin light chain kinase), and (v) control by dephosphorylation by kinases and phosphorylases (e.g., Cdc2 kinase).]
28.How would you generate prematurely condensed chromosomes. [Fuse interphase cells with those in mitosis.]
29.How would you demonstrate that a cell in G2 phase is unable to replicate its chromosomes unless it passes through mitosis? [Synchronize cells at different stages of the cell cycle, fuse, and monitor replication capacity of the different nuclei in the heterokaryon using [3H]thymidine and autoradiography. G1 x S fusions: both nuclei replicate. S x S fusions: both nuclei replicate. G2 x S fusions: G2 nucleus does not replicate.]
30.Illustrate how MPF was discovered.
31.How was cyclin discovered? [Grow fertilized sea urchin eggs in [35S]met, remove samples every 10 min, run proteins on gel. Autoradiography showed that most bands became progressively more intense as progressively more protein was made; but, two bands (i.e., cyclin A and B) first became more intense like the others but then disappeared during each mitosis to reappear during the next interphase.]
32.How were cdc mutants of budding yeast originally isolated? [A bud grows out of the mother cell prior to cell division. Screen temperature-sensitive lethal mutants for those that arrest in the cycle just after the bud has formed.]
33.Illustrate the life cycles of budding and fission yeast.
34.Illustrate the role that ORC plays in the formation of the pre-replication complex.
35.What is the phenotype of cdc2- mutants in fission yeast? [Lack the G2 checkpoint, and continue to grow into enormous cells without entering mitosis.]
36.Make notes on the role of the anaphase promoting complex (APC). [Cover: Targeting key regulators like cyclin B for proteolysis, role of ubiquitin, destruction box, proteasome, Cdc20/Fizzy. APC promotes separation of sister chromatids - roles Mad, Bub1, Bub3, APCCdc20, Pds1, Esp1, and cohesin.]
37.How was platelet-derived growth factor discovered? [Fibroblasts grow in media supplemented with serum, but not plasma. This suggested that platelets - which release their contents on clotting - might contain a growth factor, and fractionation led to the identification of PDGF.]
38.How was the START checkpoint discovered in mammalian cells? [Time-lapse cinematography and a brief deprivation of serum. Film cells for more than a cell cycle, withdraw serum for 2 h and re-add, and continue filming for another generation; trace cells back on the film to the mitosis that gave them birth. All cells that were within 3.5 h of mitosis when serum was withdrawn take an extra 8-10 h to reach the next mitosis, whereas those that were 3.5 h beyond mitosis continue unchecked to the next.]
39.How can 'density-dependent inhibition of cell division' be detected? [Plate mammalian cells in a tissue-culture dish at a low density - cells attach and divide; they continue to divide until they form a confluent monolayer. But after plating at a high density, cells attach but do not divide.]
40.Distinguish between hypertrophy and hyperplasia. [Hypertrophy: increase in cell size (e.g., cells of skeletal muscle in response to exercise). Hyperplasia: increase in cell number resulting from cell division (e.g., endometrial glands during the menstrual cycle).]
Deranged cycles and cancer
41.What evidence suggested that cancer had a genetic basis? [The clonality of tumors, and the fact that mutagens are carcinogens.]
42.What is a 'Philadelphia' chromosome? [Cells of patients with chronic myelogenous leukemia often contain a 'Philadelphia' chromosome created by translocation between the long arms of chromosomes 9 and 22. The translocation fuses the 5' end of the bcr gene to the 3' end of the abl gene (originally on chromosomes 22 and 9, respectively) to generate a new gene. As a result, the Abl tyrosine kinase is inappropriately expressed, and this drives the (neoplastic) proliferation of a clone of hemopoietic cells in the bone marrow.]
43.What is meant by the 'transformation' of a mammalian cell, and what are the properties of such cells? [Transformation: conversion of contact-inhibited cells (which grow to confluency and then stop dividing, and which are not usually tumorigenic) to those that continue to divide to give piled-up colonies (and which are often tumorigenic). Properties: (i) decreased growth factor requirements, (ii) inability to enter G0, (iii) loss of anchorage-dependence of growth and contact inhibition of movement, a changed cell morphology, and an altered microfilament network, (iv) alterations at the cell surface, including increased mobility of surface proteins, increased glucose transport, and reduced levels of fibronectin, (v) altered gene transcription, (vi) immortalization, (vii) increased tumorigenicity, (viii) often have an oncogene integrated into their genome.]
44.Distinguish between an 'oncogene' and 'anti-oncogene or 'tumor-suppressor gene'. [Oncogene: A dominant (mutant) version of a normal gene of animal cells (the proto-oncogene) that releases the cell from normal controls on growth; often found in association with other mutations in tumor cells (e.g., ras). Anti-oncogene: A recessive gene encoding a regulator that must be inactivated before a cell can divide; often mutated in tumor cells (e.g., p53, Rb).]
45.How was an activated ras gene originally detected in a bladder carcinoma? [Isolate DNA, cotransfect with a selectable marker into 3T3 cells, select transformed colonies, recover selectable marker and any associated gene, check that the associated gene now transforms 3T3 at a high frequency, sequence to confirm that ras gene contains an activating mutation.]
46.Which two general approaches led to the discovery of tumor-suppressor genes? [(i) Cell fusion (malignancy behaves as recessive when malignant mouse cell is fused with a nonmalignant cell, and then chromosomes are gradually lost). (ii) Epidemiology of retinoblastoma (hereditary autosomal dominant and nonhereditary sporadic explained by mutations in two recessive genes).]
47.List the approaches being used to identify the genetic defects that underlie cancer. [Approach used for Rb, transfection assay (e.g., ras), by homology with oncogenes of RNA tumor viruses (e.g., abl), genes at sites of integration of retroviruses (e.g., Wnt-1), genes at translocation sites (e.g., myc), amplified genes (N-myc).]
48.Make notes on the T antigen of SV40 virus. [Cover: antigen expressed in cells infected with the virus, and in tumors containing integrated virus, a multifunctional 90 kD protein, role in driving infected G1 cell into S phase so virus can subvert cell's replication machinery, phosphorylation by Cdc2 kinase, interaction with p53.]
49.How was p53 discovered? [By co-precipitation with T antigen from cell extracts.]
50.Make notes on the role of p53 in tumorigenesis. [Cover: Li-Fraumeni syndrome, ~75% colorectal cancers associated with mutations in p53, protein structure (N-terminal 'transcriptional activation' domain, central DNA-binding domain, C-terminal domain), target for numerous kinases and phosphatases, role as transcription factor and in controlling cell cycle progression and apoptosis.]
51.Make notes on colorectal cancer. [Cover: Second most common cause of cancer death in USA, ~15% colorectal cancers occur in dominantly inherited patterns, familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), multi-step progression (dysplastic aberrant crypt foci, early adenomas, intermediate adenomas, late adenomas, carcinomas), involvement of APC, p53, DCC, and MMR genes.]
Apoptosis
52.What is apoptosis? [Physiological cell death, characterized by chromatin condensation, nuclear fragmentation, and cell shrinkage (contrast with pathological cell death, characterized by inflammation and necrosis).]
53.How were the Ced genes discovered? [Ced-1 and Ced-2 were recognized because mutant embryos contained refractive cells that persisted without resorption for days. Ced-3 was obtained by treating Ced-1 hermaphrodites with EMS - F2 progeny no longer contained the refractive cells.]
54.Illustrate the similarities between the death pathways of the worm and man. [Cover: Ced-9, Ced-4 and Ced-3 in the worm; Bcl-2, Apaf-1, and caspase-9 in man.]
55.Make notes on caspases. [Cover: cysteine proteases specific for aspartate, expressed as proenzymes containing three domains (NH2 regulatory domain, large and small subunit), activated by cleavage, active heterodimer of large and small subunits, recognize specific tetrapeptide and cut after asp (e.g., caspase-3 cuts and DEVD), roles (in apoptosis, normal development, killing of virally-infected cells).]
56.Illustrate how CD95L (fas ligand) activates the death pathway. [Include: CD95L, CD95, FADD, caspases.]
Overview
1.Outline the changes that occur in ploidy during meiosis in man. [A 2n cell goes through S phase to give a 4n cell; then, two successive divisions (meiosis I and II) generate haploid gametes.]
Meiosis
2.Which central features of meiosis ensure that the haploid generation receives a mixed set of genes (increasing genetic variation)? [Segments of homologous chromosomes are recombined at random, and maternal and paternal homologs are segregated semi-randomly at the first division.]
3.Illustrate the differences between meiosis and mitosis.
4.What is meant by the terms synapsis and chiasma, as applied to chromosomes? [Synapsis: the close apposition, or pairing, of homologous chromosomes. Chiasma: the junction (resulting from recombination between maternal and paternal chromatids) that results between non-sister chromatids at the first diplotene of meiosis.]
5.List the different stages of meiosis and the functions associated with them. [Prophase I (reductional division): leptotene (chromosomes condense and search for their partners), zygotene (homologs align), pachytene (tight alignment), diplotene (unpairing), and diakinesis (moving apart). Metaphase I is followed by an interphase, and then metaphase II (equational division).]
6.What is a synaptonemal complex and a recombination nodule? [Synaptonemal complex: A ladder-like structure (length >100 nm) seen in the electron microscope that lies between synapsed chromosomes and which is intimately associated with crossing over; it consists of a central plate connected by filaments to two lateral plates that lie along the flanking chromosomes. Recombination nodule: Small structure (~100 nm diameter) attached to the synaptonemal complex that may be site of recombination.]
7.How do human X and Y chromosomes pair during meiosis? [The two chromosomes share a small region of homology - the pseudoautosomal region - at their ends.]
8.What are the first and second polar bodies (in the context of oogenesis in a mammal)? [The first of the two meiotic divisions leads to the formation of a small (first) polar body and a larger cell; the latter then goes through the second meiotic division to give the egg and the second polar body.]
Recombination
9.Recombination can be classified into three main types; what are they? [General, site-specific, and illegitimate.]
10.List the general principles involved in recombination. [Cover: Two homologous DNA duplexes align and cross-over (double helices are broken, a broken end from one partner is joined to that of the other to reform intact duplexes), initiation depends on a homology search, the cross-over usually gives a staggered (heteroduplex) joint, cleavage and rejoining are generally precise, flanking sequences can be shuffled in new combinations, reciprocal and non-reciprocal exchange.]
11.Illustrate how resolution of a 'Holliday' junction can give chromosomes that have exchanged information, and others that have not.
12.Illustrate branch migration in a three-stranded molecule and a 'Holliday' junction.
13.Make notes on the bacterial RecA protein. [Cover: The RecBCD pathway in E. coli, reconstitution of recombination in vitro using RecBCD and RecA (38 kD monomer); Rec A performs various roles - it aggregates DNA molecules by binding cooperatively ( 5'->3') to give a nucleoprotein filament with extended DNA (18.6 bp per turn, 0.95 nm between base pairs), and it catalyzes branch migration (5'->3' direction with respect to the incoming single-stranded DNA).]
14.Illustrate the changes in gene position that might occur during gene conversion in yeast.
Chromosome pairing
15.Give some examples of non-meiotic pairing between homologous chromosomes. [Yeast: most chromosomal loci. Drosophila: histone loci pair between embryonic cycles 12 and 14, almost all loci pair in polytene chromosomes. Man: repeated 45S rRNA genes.]
16.Illustrate how transcriptional activity might underlie chromosome pairing.