What is the relationship of DNA, a chromosome, and chromatin? | Socratic
Although the numbers and sizes of chromosomes vary considerably The DNA of eukaryotic cells is tightly bound to small basic proteins (histones) that. Book / Writing tools / Some questions for review (with answers) Roughly, when were cells, nuclei, chromosomes, and DNA discovered? .. Illustrate the relationship between the membranes of the endoplasmic reticulum and the nuclear. In one sentance, the relationship can be summed up as: "Chromatin is the complex of DNA and protein that makes up chromosomes." To expand further: DNA is.
These results suggested that the binding of proteins to DNA in chromatin protects regions of the DNA from nuclease digestion, so that the enzyme can attack DNA only at sites separated by approximately base pairs.
Consistent with this notion, electron microscopy revealed that chromatin fibers have a beaded appearance, with the beads spaced at intervals of approximately base pairs. Thus, both the nuclease digestion and the electron microscopic studies suggested that chromatin is composed of repeating base-pair units, which were called nucleosomes. Nonhistone proteins bind to the linker DNA between nucleosome core particles. B Gel electrophoresis of DNA fragments more More extensive digestion of chromatin with micrococcal nuclease was found to yield particles called nucleosome core particles that correspond to the beads visible by electron microscopy.
Detailed analysis of these particles has shown that they contain base pairs of DNA wrapped 1. One molecule of the fifth histone, H1, is bound to the DNA as it enters each nucleosome core particle. This forms a chromatin subunit known as a chromatosomewhich consists of base pairs of DNA wrapped around the histone core and held in place by H1 a linker histone.
A The nucleosome core particle consists of base pairs of DNA wrapped 1. A chromatosome contains two full turns of DNA base more The packaging of DNA with histones yields a chromatin fiber approximately 10 nm in diameter that is composed of chromatosomes separated by linker DNA segments averaging about 80 base pairs in length Figure 4. In the electron microscope, this nm fiber has the beaded appearance that suggested the nucleosome model.
Packaging of DNA into such a nm chromatin fiber shortens its length approximately sixfold. The chromatin can then be further condensed by coiling into nm fibers, the structure of which still remains to be determined. Interactions between histone H1 molecules appear to play an important role in this stage of chromatin condensation. The packaging of DNA into nucleosomes yields a chromatin fiber approximately 10 nm in diameter.
The chromatin is further condensed by coiling into a nm fiber, containing about six nucleosomes per turn. Photographs courtesy of Ada more The extent of chromatin condensation varies during the life cycle of the cell. In interphase nondividing cells, most of the chromatin called euchromatin is relatively decondensed and distributed throughout the nucleus Figure 4. During this period of the cell cycle, genes are transcribed and the DNA is replicated in preparation for cell division.
Most of the euchromatin in interphase nuclei appears to be in the form of nm fibers, organized into large loops containing approximately 50 to kb of DNA. Chromatin structure is thus intimately linked to the control of gene expression in eukaryotes, as will be discussed in Chapter 6. Electron micrograph of an interphase nucleus. The euchromatin is distributed throughout the nucleus.
Chromosomes and Chromatin - The Cell - NCBI Bookshelf
The hetero-chromatin is indicated by arrowheads, and the nucleolus by an arrow. What is the evidence for this structure? How can the lengths of DNA in the nucleosome, and between nucleosomes, be determined?
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. Describe the evidence for and against the existence in vivo of a 'solenoid'. What is the evidence that the chromatin fiber is organized into loops in the interphase nucleus?
Go onto discuss the analysis of residual fragments, and how the results suggest that attachments and so loops change continually. Outline the path of DNA duplexes in the 'lampbrush' chromosomes of amphibia. Describe how the contour length of a chromatin loop can be measured. 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? 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. What is an 'insulator', and how were they discovered? Insulators are able to prevent such silencing. They are discovered by inserting test sequences next to a reporter gene e. What is 'position effect variegation', and how was it discovered? 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 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.
Review questions, with answers (Peter R Cook's book)
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. How is the 'territory' occupied by a chromosome during interphase identified?
What is the 'Rabl' orientation? What major factors determine the location of a chromosome within the interphase nucleus?
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? In this case, spreads of mitotic chromosomes or interphase cells growing on a coverslip are fixed, and their DNA heated to denature it.
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. Why has it been so difficult to observe the nucleoskeleton, and what are its constituents?
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. What are their roles? PIKA polymorphic interphase karyosomal association domain: Roles of all unknown.
What is an autosome, chromatid, and karyotype? Outline the essential structural features shared by all eukaryotic chromosomes. What is an 'autonomously-replicating sequence' ARS?
How was the first one identified in yeast? DNA sequence that enables a circular plasmid lacking an origin to replicate in yeast cells, usually equivalent to origin of replication. 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.
What is a YAC, and how is it used? What are chromosome bands, and how are they produced? 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.
Outline the 'scaffold' model for chromosome structure, and what is the evidence for it? An axial 'scaffold' containing topoisomerase II forms an underlying structure to which chromatin fibres are attached in loops. Images of 'scaffolds' isolated from nuclei provide the best evidence for this kind of model; DNA loops are seen attached to the central axis.
Outline the path of DNA duplexes in a 'polytene' chromosome. Outline the principles involved in eukaryotic DNA synthesis. How would you demonstrate that active DNA polymerases are fixed to an underlying structure in the nucleus? Outline the different approaches used to label sites of DNA synthesis in eukaryotic nuclei, and the difficulties associated with each one.
By autoradiography with [3H]thymidine: By immunolabeling after incubation with Br-dU: Details of factories best seen after removing most chromatin. How were replication factories imaged in B. What is the unwinding problem, and how might it be solved in theory and in practice? Outline the reactions performed by type I and II topoisomerases.
Therefore, a special RNA polymerase - the primase - is used to make a short RNA chain the primer of nucleotides, so a DNA polymerase can take over to catalyze addition of a deoxynucleotide onto the 3' OH of the primer. Draw template and nascent strands at a replication fork; label the 5' and 3' ends, and the leading and lagging strands.
What are Okazaki fragments, and how were they discovered? Using T4 bacteriophage; infected bacteria were incubated at 8C for 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, 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.
Draw a diagram illustrating the history i. Mark the 5' and 3'ends. List as many different enzymic activities required during DNA synthesis as you can. What is a proofreading activity? How is histone synthesis coupled to DNA synthesis? CAF-1 and Nap-1 facilitate assembly into nucleosomes. Describe the structure of the origin of replication in E. What information does DNA fiber autoradiography yield? How many origins does a typical human chromosome of Mbp contain?
Outline the structure of the origin of SV40 virus. DNA sequence that enables circular plasmid lacking origin to replicate in yeast cells, usually equivalent to an origin of replication. The first ARS was obtained as follows. Yeast mutants lacking the LEU gene cannot form colonies without added leucine.
Outline the structure of a typical yeast origin. Outline two methods for mapping origins. 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.
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.
How would you show that the same origins are used in a mammalian cell during successive cell cycles? If the same origins are used during successive S phases, regions labelled with one color will also be labeled with the second. Outline the problem associated with replicating the ends of a chromosome, and some solutions. 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.
Outline the properties of telomerase. Outline the basic principles involved in eukaryotic RNA synthesis. How would you determine which parts of the genome are transcribed? How would you demonstrate that active RNA polymerases are fixed to an underlying structure in the nucleus?
How would you localize sites of RNA synthesis in eukaryotic nuclei? Describe the properties of the bacterial RNA polymerase. What are the untwining and supercoiling problems, and how are they resolved? Supercoiling problem and solution: How was it demonstrated that attached RNA polymerases can work?
How would you measure the pulling power of a polymerase? Roughly, what force acting on DNA can stall a polymerase, strip a nucleosome from the template, and break the duplex?
Draw the general structures of the transcription units transcribed by the three eukaryotic polymerases. How is a 'Miller' spread prepared, and illustrate the appearance of a spread containing some ribosomal cistrons? Why is it so difficult to estimate the number of active polymerases in the cell? Roughly, how many polymerases are associated with a typical transcription unit? What is a DNA 'microarray? 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. Fibrillar center store with active polymerases on the surfacedense fibrillar component nascent transcriptsgranular component maturation site. 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.
How would you label nascent transcripts in transcription factories?
What is 'transcriptional interference', and how was it discovered? 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. What are the main modifications made to a polymerase II transcript after it has been made?
Draw the structure of a typical cap found at the 5' end of a message. How can caps be isolated? Which radiolabels would you use to monitor capping? What is the role of the cap? What sequence motifs in RNA trigger polyadenylation in mammals? What factors are required to constitute polyadenylation in vitro?
How are chromosomes related to chromatin?
What is the role of the poly A tail? What is an intron and exon? How were introns discovered? Why do you think introns evolved? Draw the structure of the nucleotide at the branch-point in the lariat. How would you demonstrate that some splicing occurs co-transcriptionally? What is 'nonsense mediated decay' NMDand how was it discovered? List the natural agents that commonly cause damage in our DNA.
List the lesions most commonly found in our DNA. What makes it so difficult to study the repair of damage in DNA?
What methods have led to our understanding of repair mechanisms? Make notes on repair defects and human disease. What are the basic biochemical steps found in many repair pathways? What are the three main repair pathways found in man, and what makes them distinctive? Give an example of the events occurring during 'direct repair'. 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.
Outline the events occurring during 'base excision repair'. 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.
Outline the events occurring during 'nucleotide excision repair'.Genes, DNA and Chromosomes explained
One major pathway operates throughout the genome, another is coupled to transcription. How was the size of the 'patch' of DNA removed during nucleotide excision repair determined? 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 limits of the repaired patch are mapped relative to the known positions of the oligonucleotide and restriction sites. Outline the events occurring during 'mismatch repair'. Human cells contain homologs of these proteins, and some of these homologs e.
What are the three major damage response systems in bacteria? The adaptive response to alkylating agents: The adaptive response to oxidative stress: 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? 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.
Outline the evidence that some repair is coupled to transcription? What are the main consequences of introducing damage into the DNA of mammalian cells? If cells with damaged DNA continue to divide, mutations become fixed, and cancer may result. Outline the central features of the somatic mutation theory of cancer, and the evidence for it. Differentiate between positive and negative control mechanisms in bacteria.
Outline the effects that occur when tryptophan switches off expression of the trp operon. 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.
How does the catabolite activator protein promote expression of catabolic enzymes in bacteria? 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.
Draw cartoons illustrating how the lac and lambda repressors regulate the activity of their respective operons. How was it demonstrated that GAL4 has two distinct functional domains? How would you test the totipotency of the nucleus in a frog fibroblast?
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.
How was 'Dolly' the lamb cloned?