![Transcription factories in a Hela cell [from Cook PR (1999) Science 284, 1790]](../images/pombo.png)
Go to the same questions with answers.
Overview of the cell nucleus
1.Roughly, when were cells, nuclei, chromosomes, and DNA discovered?
2.When did Mendel, and Watson and Crick publish their seminal works?
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?
4.What is the approximate contour length of DNA of a typical human chromosome containing 100 Mbp? What is (roughly) the length of a mitotic chromosome?
5.What is meant by the resolution of a microscope, and what is the resolution of the light and electron microscope?
6.What processes are involved in preparing an image like that of the histological section shown in Fig. 1-1?
7.What is meant by the terms fixation, embedding, and staining (as applied to a histological section like that illustrated in Fig. 1-1)?
8.What are the differences between freeze-fracture, freeze-drying, freeze-etching, and freeze-substitution?
9.What is the green fluorescent protein, and how does it fluoresce?
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? How was the data obtained?
12.Roughly, how far apart are molecules when in solution at 1 μM (and 1 mM)?
Structures of nucleic acids
13.What are the components of a nucleotide?
14.Draw the structure of a named nucleotide.
15.What are the chemical differences between DNA and RNA?
16.What are the purines and pyrimidines found in DNA and 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?
20.What do we mean when we say that the strands in a DNA duplex are 'anti-parallel'?
21.How many base pairs are there in one turn of the B form of DNA? What is the angular rotation (about the helical axis) between each base pair?
22.What form of DNA exists in solution in an isotonic buffer? How was this determined?
23.What are the major and minor grooves in DNA?
24.What is meant by melting (denaturing) DNA? How can this be achieved?
25.What is the melting temperature of DNA, and how can it be determined?
26.What is the rate-limiting step during reannealing of two complementary DNA strands? What equation governs the rate of reannealing?
27.What properties of DNA are essential for its role as a carrier of genetic information?
28.Outline how genetic information can flow between DNA, RNA, and protein.
29.How many base-pairs does a typical human chromosome contain? What is the contour length of such a DNA molecule?
30.What is the 'packing problem'?
31.How was it shown that a single DNA molecule runs the length of a eukaryotic chromosome?
32.How are DNA molecules of different sizes separated during gel electrophoresis?
33.What is pulsed-field electrophoresis?
34.Why are binding sites for proteins often phased every 10 bp along a DNA duplex?
35.How can a DNA duplex bend?
36.What is DNA supercoiling?
37.What does a DNA topoisomerase (type I and II) do?
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?
40.What equation describes the helical windings of a strand in a DNA molecule?
41.What is a 'restrained' supercoil? Provide an example.
42.How does the structure of RNA differ from that of DNA?
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.
47.What is DNA 'footprinting'?
48.Why are DNA-binding proteins often dimers?
49.Write out a palindromic DNA sequence.
50.Make notes on some DNA-binding motifs found in proteins.
Making large structures
51.What are the general rules that govern the biosynthesis of all polymers? What advantages does a strategy based on these rules provide?
52.Why are so many biopolymers helical?
53.What is a molecular 'chaperone'?
54.Why do biologists use extracts of Xenopus eggs so frequently?
55.What is a 'minichromosome'? How can it be made in vitro, and what does it contain?
56.What properties of a protein ensure that it is localized to a particular part of a cell?
57.What is meant by 'tensegrity'? Give an example of a tensegrity structure, and what advantages do tensegrity structures have over more conventional ones?
58.What determines the position of a nucleus in a cell?
59.Why are nuclei often round?
Some evolutionary considerations
60.Outline Darwin's theory of natural selection.
61.What three generalizations underpin Darwin's theory of evolution by natural selection.
62.Make brief notes on pseudo-genes, junk DNA, retrotransposons, satellites, LINEs, and alu repeats.
63.What is a 'Cot curve'? How is the Cot1/2 related to genome complexity?
64.Roughly, how much DNA is contained in the haploid human genome? How was this determined?
65.What is the size of a typical gene and exon in the human genome? How frequently are genes spaced along the chromosome?
66.Roughly, what is the genome size and gene number of E. coli, S. cerevisiae, C. elegans, D. melanogaster, and H. sapiens?
67.What evidence indicates that most genes in the fly are expressed in many different tissues?
68.Briefly describe the extra-nuclear DNA found in mammalian cells.
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?
70.Describe the yeast 'two-hybrid' system and its uses.
71.What is FRET, and how is it used?
72.What is meant by 'epistasis', and how is it used?
73.How does a bacterium swim toward a source of food?
74.Make short notes on why you think nuclei evolved.
75.What is 'endosymbiosis'?
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.
2.What are the stages of mitosis?
3.How are 'spreads' of metaphase chromosomes prepared?
The nuclear membrane
4.Illustrate the relationship between the membranes of the endoplasmic reticulum and the nuclear envelope.
5.What is the nuclear lamina?
6.Describe the general structure of intermediate filaments (IFs). How do the lamins differ from the other IFs?
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?
9.Outline how proteins of >60 kD enter the nucleus.
10.What are nuclear localization signals (NLSs), nuclear export signals (NESs), and nuclear retention signals (NRSs)? How are such signals identified?
11.Describe how autoradiography can be used to identify which cells in a population are in S phase.
12.How is RNA exported through the pore? Provide experimental details of how the different mechanisms were uncovered.
13.Is the nuclear membrane an ion barrier?
The nucleolus
14.What are the different types of ribosomal RNA, and which polymerases are responsible for their synthesis?
15.What are the main subcompartments within nucleoli, and what is their role?
16.Describe the organization of rDNA sequences in the human genome.
Packaging chromatin during interphase
17.What is the 'packing problem', and how is it solved?
18.Why is it difficult to analyze nuclei and chromatin in isotonic buffers?
19.Describe how nuclei, 'matrices', 'scaffolds', and 'nucleoids' are usually isolated.
20.Describe how 'matrix attachment regions' and 'scaffold attachment regions' are identified.
21.What are the 'nucleosomal' 'core' histones?
22.Outline the general structure of the nucleosome. What is the evidence for this structure?
23.How can the lengths of DNA in the nucleosome, and between nucleosomes, be determined?
24.Describe the evidence for and against the existence in vivo of a 'solenoid'.
25.What is the evidence that the chromatin fiber is organized into loops in the interphase nucleus?
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.
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?
29.What is an 'insulator', and how were they discovered?
30.What is 'position effect variegation', and how was it discovered?
31.How is the 'territory' occupied by a chromosome during interphase identified?
32.What is the 'Rabl' orientation?
33.What major factors determine the location of a chromosome within the interphase nucleus?
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?
Nucleoskeletons and nuclear sub-compartments
35.Why has it been so difficult to observe the nucleoskeleton, and what are its constituents?
36.Make brief notes on nuclear bodies and dots, coiled (or Cajal) bodies, PML bodies, and PIKA domains. What are their roles?
Chromosomes
37.What is an autosome, chromatid, and karyotype?
38.Outline the essential structural features shared by all eukaryotic chromosomes.
39.What is an 'autonomously-replicating sequence' (ARS)? How was the first one identified in yeast?
40.How would you show that a DNA duplex (~1 kbp) contained a region that could function as a centromere (or telomere) in yeast?
41.What is a YAC, and how is it used?
42.What are chromosome bands, and how are they produced?
43.Outline the 'scaffold' model for chromosome structure, and what is the evidence for it?
44.Outline the path of DNA duplexes in a 'polytene' chromosome.
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.
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?
Replication factories
5.Outline the different approaches used to label sites of DNA synthesis in eukaryotic nuclei, and the difficulties associated with each one.
6.How were replication factories imaged in B. subtilis?
The mechanics of synthesis at the fork
7.What is the unwinding problem, and how might it be solved in theory and in practice?
8.Outline the reactions performed by type I and II topoisomerases.
9.Outline the role of DNA helicases in DNA synthesis.
10.Outline the role of single-strand binding (SSB) proteins in DNA synthesis.
11.Outline the role of RNA primers in DNA synthesis.
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?
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.
16.What is a proofreading activity?
17.How is histone synthesis coupled to DNA synthesis?
The initiation of synthesis
18.Describe the structure of the origin of replication in E. coli.
19.What information does DNA fiber autoradiography yield?
20.How many origins does a typical human chromosome of 100 Mbp contain?
21.Outline the structure of the origin of SV40 virus.
22.What is an 'autonomously-replicating sequence' (ARS)? How was the first one identified in yeast?
23.Outline the structure of a typical yeast origin.
24.Outline two methods for mapping origins.
25.How would you show that the same origins are used in a mammalian cell during successive cell cycles?
26.When during S phase is the DNA in R and G bands replicated?
Replicating ends
27.Outline the problem associated with replicating the ends of a chromosome, and some solutions.
28.Outline the properties of telomerase.
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.
3.How would you determine which parts of the genome are transcribed?
Tracking versus immobile RNA polymerases
4.How would you demonstrate that active RNA polymerases are fixed to an underlying structure in the nucleus?
5.How would you localize sites of RNA synthesis in eukaryotic nuclei?
6.Describe the properties of the bacterial RNA polymerase.
7.What are the untwining and supercoiling problems, and how are they resolved?
8.How was it demonstrated that attached RNA polymerases can work?
9.How would you measure the pulling power of a polymerase?
10.Roughly, what force acting on DNA can stall a polymerase, strip a nucleosome from the template, and break the duplex?
The three kinds of eukaryotic RNA polymerase
11.How can the activities of RNA polymerases I, II, and III, be distinguished?
12.Which classes of RNA do RNA polymerases I, II, and III make?
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?
15.Distinguish between: the core enzyme of RNA polymerase II and the holoenzyme, general and specific transcription factors, initiation and elongation factors.
16.Why is it so difficult to estimate the number of active polymerases in the cell?
17.Roughly, how many polymerases are associated with a typical transcription unit?
18.What is a DNA 'microarray?' Give an example of how one might be used.
Transcription factories
19.What are the main subcompartments within nucleoli, and what is their role?
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?
22.What is 'transcriptional interference', and how was it discovered?
Processing and transport of polymerase II transcripts
23.What are the main modifications made to a polymerase II transcript after it has been made?
24.Draw the structure of a typical cap found at the 5' end of a message.
25.How can caps be isolated?
26.Which radiolabels would you use to monitor capping?
27.What is the role of the cap?
28.What sequence motifs in RNA trigger polyadenylation in mammals?
29.What factors are required to constitute polyadenylation in vitro?
30.What is the role of the poly(A) tail?
31.What is an intron and exon?
32.How were introns discovered?
33.Why do you think introns evolved?
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?
36.What is 'nonsense mediated decay' (NMD), and how was it discovered?
The need for repair of damage
1.List the natural agents that commonly cause damage in our DNA.
2.List the lesions most commonly found in our DNA.
3.What makes it so difficult to study the repair of damage in DNA?
Some experimental approaches
4.What methods have led to our understanding of repair mechanisms?
5.Make notes on repair defects and human disease.
Types of repair
6.What are the basic biochemical steps found in many repair pathways?
7.What are the three main repair pathways found in man, and what makes them distinctive?
8.Give an example of the events occurring during 'direct repair'.
9.Outline the events occurring during 'base excision repair'.
10.Outline the events occurring during 'nucleotide excision repair'.
11.How was the size of the 'patch' of DNA removed during nucleotide excision repair determined?
12.Outline the events occurring during 'mismatch repair'.
13.What are the three major damage response systems in bacteria?
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?
15.Outline the evidence that some repair is coupled to transcription?
Some consequences of inefficient repair
16.What are the main consequences of introducing damage into the DNA of mammalian cells?
17.Outline the central features of the somatic mutation theory of cancer,
and the evidence for it.
6: REGULATION OF GENE EXPRESSION
Simple regulatory circuits in bacteria and yeast
1.Differentiate between positive and negative control mechanisms in bacteria.
2.Outline the effects that occur when tryptophan switches off expression of the trp operon.
3.How does the catabolite activator protein promote expression of catabolic enzymes in bacteria?
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?
Principles of eukaryotic gene regulation
6.How would you test the totipotency of the nucleus in a frog fibroblast?
7.How was 'Dolly' the lamb cloned?
8.How were the rearrangements that occur in immunoglobulin genes during the development of B cells discovered?
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?
11.List some examples of non-genic transcripts that control gene activity.
12.How do restriction enzymes get their name?
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?
15.Draw a cartoon illustrating the way alternative splicing of sxl transcripts determines sex in Drosophila.
16.What is RNA interference?
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)?
19.Give an example of an experiment showing that differential expression of a gene can require continuous regulation?
20.What is 'subtractive' hybridization' of a cDNA library? Outline how it was used to identify myoD.
21.How was it shown that the activity of a eukaryotic gene can be regulated over eight 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.
23.How would you distinguish between DNase 'sensitive' and 'hypersensitive' regions in chromatin?
24.Where are histones acetylated?
25.List the different covalent modifications of histones.
26.How are chromatin remodeling machines detected?
27.List some chromatin remodeling machines.
Regulation at the level of the loop
28.What is an enhancer and locus control region (LCR)?
29.How would you perform a 'run-on' transcription reaction, and for what is it used?
Heterochromatin
30.List some properties of heterochromatin.
31.What is meant by the term 'silencing' in yeast?
32.What is a 'CpG island'?
Establishing and inheriting patterns of expression
33.Illustrate how the activity of rDNA genes can be inherited through mitosis.
34.How would you demonstrate the effects of a maternal effect gene in Drosophila?
Overview
1.List the phases of the mammalian cell cycle.
2.How was the cell cycle discovered?
3.How would you obtain mammalian cells synchronized at different phases of the cell cycle?
Mitosis
4.Define mitosis.
5.What is cytokinesis?
6.What are the stages of mitosis?
7.Illustrate the changes that occur to a chromosome as it progresses around the cell cycle.
8.Make notes on microtubules.
9.What is the centrosome, or microtubule organizing center?
10.What is the centriole, and what are its roles?
11.Illustrate the centrosome cycle in an animal cell.
12.What is a kinetochore?
13.List the different types of microtubules found in the spindle at metaphase.
14.Draw a spindle with attached metaphase chromosomes; mark the different kinds of microtubules.
15.List the requirements for exact segregation of chromosomes.
16.List the roles of centromeres during mitosis.
17.How would you classify centromeres?
18.Outline the structure of a centromere of S. cerevisiae. Which of its elements are essential?
19.Outline the structure of a human centromere.
20.How did Nicklas and Koch (1960) show that the tension generated by conflicting forces guides chromosome pairs toward the correct bipolar alignment?
21.What are the two classes of microtubule motor?
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.
Regulation of the cell cycle
24.In which phases of the cell cycle are the major checkpoints in budding and fission yeast?
25.What are the two major components of maturation promoting factor (and of the G2 checkpoint machinery in fission yeast)?
26.Outline how adrenaline regulates glycogen levels in skeletal muscle.
27.Outline the various ways that the catalytic activity of the different members of the human 'kinase' family are regulated.
28.How would you generate prematurely condensed chromosomes.
29.How would you demonstrate that a cell in G2 phase is unable to replicate its chromosomes unless it passes through mitosis?
30.Illustrate how MPF was discovered.
31.How was cyclin discovered?
32.How were cdc mutants of budding yeast originally isolated?
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?
36.Make notes on the role of the anaphase promoting complex (APC).
37.How was platelet-derived growth factor discovered?
38.How was the START checkpoint discovered in mammalian cells?
39.How can 'density-dependent inhibition of cell division' be detected?
40.Distinguish between hypertrophy and hyperplasia.
Deranged cycles and cancer
41.What evidence suggested that cancer had a genetic basis?
42.What is a 'Philadelphia' chromosome?
43.What is meant by the 'transformation' of a mammalian cell, and what are the properties of such cells?
44.Distinguish between an 'oncogene' and 'anti-oncogene or 'tumor-suppressor gene'.
45.How was an activated ras gene originally detected in a bladder carcinoma?
46.Which two general approaches led to the discovery of tumor-suppressor genes?
47.List the approaches being used to identify the genetic defects that underlie cancer.
48.Make notes on the T antigen of SV40 virus.
49.How was p53 discovered?
50.Make notes on the role of p53 in tumorigenesis.
51.Make notes on colorectal cancer.
Apoptosis
52.What is apoptosis?
53.How were the Ced genes discovered?
54.Illustrate the similarities between the death pathways of the worm and man.
55.Make notes on caspases.
56.Illustrate how CD95L (fas ligand) activates the death pathway.
Overview
1.Outline the changes that occur in ploidy during meiosis in man.
Meiosis
2.Which central features of meiosis ensure that the haploid generation receives a mixed set of genes (increasing genetic variation)?
3.Illustrate the differences between meiosis and mitosis.
4.What is meant by the terms synapsis and chiasma, as applied to chromosomes?
5.List the different stages of meiosis and the functions associated with them.
6.What is a synaptonemal complex and a recombination nodule?
7.How do human X and Y chromosomes pair during meiosis?
8.What are the first and second polar bodies (in the context of oogenesis in a mammal)?
Recombination
9.Recombination can be classified into three main types; what are they?
10.List the general principles involved in recombination.
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.
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.
16.Illustrate how transcriptional activity might underlie chromosome pairing.