The Nucleus PDF

Exam 2 results The Nucleus Lesson 15  The nuclear envelope defines the nucleus The nucleus contains the cell's genetic material, composed of DNA organized into chromosomes. It is surrounded by two membrane bilayers, the inner and outer nuclear membrane The outer membrane is continu...

Exam 2 results The Nucleus Lesson 15  The nuclear envelope defines the nucleus The nucleus contains the cell's genetic material, composed of DNA organized into chromosomes. It is surrounded by two membrane bilayers, the inner and outer nuclear membrane The outer membrane is continuous with the ER (both compartments share the same lumen). The nucleus is studded with nuclear pores that allow proteins and mRNA to move between the nucleus and cytoplasm.  The nuclear lamins form a basket-like network of filaments that protects the nucleus from mechanical forces Nuclear lamins are intermediate filaments present in the nucleus of all eukaryotic cells Nuclear lamins form a tough network of filaments within the nucleus just underneath the nuclear membrane (B) shows a TEM image of the inner surface of the nuclear membrane; the nuclear lamins are visible as a lattice-like 2- dimensional meshwork Lamins protect the nucleus from mechanical forces Lamins also provide an attachment site for chromosomes during interphase and likely contribute to gene expression through mechanisms we do not yet understand  Mutations in lamins are associated with Hutchinson– Gilford syndrome (aka progeria) Evidence that nuclear lamins play an important role in gene regulation has come from studying a disease called progeria The image on the left is of an 11-year-old girl suffering from progeria Progeria afflicts people with symptoms of premature aging: it causes hair loss, wrinkled skin, atherosclerosis, blindness, kidney failure cardiovascular disease People with this disease rarely survive into their teenage years Some forms of progeria are caused by mutation of a single amino acid residue in one of our lamin genes People with this mutation exhibit altered patterns of gene  expression that could cause these symptoms How much of what we consider to be the ”normal” consequences of aging is due to changes in gene expression over our lifetime? Could insight gained from studying progeria patients tell us more about normal aging? Nuclei of progeria patients exhibit abnormal shapes that is consistent with the role for lamins in maintaining nuclear shape Most genes contain information to make proteins  The structure of a eukaryotic protein-coding gene The structure of a eukaryotic protein-coding gene. The red DNA segments represent the protein coding region that specifies the amino acid sequence. Regulatory sequences (yellow and blue) can affect transcriptional and translational regulation of gene expression. Introns (grey) are non-coding sequences that are transcribed but spliced from the mRNA before it is translated. The genomes of many eukaryotes carry an excess of interspersed non-coding “junk” DNA  Chromosome duplication and segregation occurs via an ordered cell cycle in proliferating cells During interphase, the cell expresses many of its genes, and— during part of this phase—it duplicates its chromosomes. Once chromosome duplication is complete, the cell can enter M phase, during which nuclear division, or mitosis, occurs. In mitosis, the duplicated chromosomes condense, gene expression largely ceases, the nuclear envelope breaks down, and the mitotic spindle forms from microtubules and other proteins. The mitotic spindle then captures the condensed chromosomes. Next, one complete set of chromosomes is pulled to each end of the cell, and a nuclear envelope forms around each chromosome set. In the final step of M phase, the cell divides to produce two daughter cells. Only two different chromosomes are shown here for simplicity. Fluorescence in situ hybridization (FISH) permits visualization of chromosomes by microscopy Fluorescent molecules  Eukaryotic DNA is packaged into multiple chromosomes The chromosomes shown here were isolated from a cell undergoing nuclear division (mitosis) and are in a highly compact (condensed) state. Chromosome painting is done by exposing the chromosomes to a collection of single-stranded DNA molecules coupled with a combination of fluorescent dyes (see next slide). For example, single-stranded DNA molecules that match sequences in chromosome 1 are labeled with one specific dye combination, those that match sequences in chromosome 2 with another, and so on. (A) Micrograph showing the array of chromosomes as they originally spilled from the lysed cell. (B) The same chromosomes are artificially lined up in their numerical order. This arrangement of the full chromosome set is called a karyotype.  Three DNA sequence elements are needed to produce a eukaryotic chromosome that can be duplicated and then segregated at mitosis Each chromosome has multiple origins of replication, one centromere and two telomeres. The sequence of events a typical chromosome follows during the cell cycle is shown schematically. The DNA replicates in interphase, beginning at the origins of replication and proceeding bidirectionally from each origin along the chromosome to the telomeres. Telomeres contain DNA sequences that allow for the complete replication of chromosome ends. In M phase, the centromere attaches the compact, duplicated chromosomes to the mitotic spindle so that one copy will be distributed to each daughter cell when the cell divides. Before cell division, the centromere also helps to hold the duplicated chromosomes together until they are ready to be pulled apart.  Interphase chromosomes occupy distinct territories within the nucleus DNA probes coupled with different fluorescent markers paint individual interphase chromosomes in a human cell. (A) Viewed in a fluorescence microscope, the nucleus is filled with a patchwork of discrete colors. (B) Three sets of chromosomes (3, 5, and 11) are singled out to highlight their distinct locations. Note that pairs of homologous chromosomes, such as the two copies of chromosome 3 (green), are not generally located in the same position.  The nucleolus is the most prominent structure in the interphase nucleus (A) Electron micrograph of a thin section through the nucleus of a human fibroblast. The nuclear envelope surrounds the nucleus. Inside the nucleus, the chromatin appears as a diffuse speckled mass; especially dense regions are called heterochromatin (dark staining). Heterochromatin contains few genes and is located mainly around the periphery of the nucleus, immediately under the nuclear envelope. The large, dark region within the nucleus is the nucleolus, which contains the genes for ribosomal RNAs. (B) Schematic illustration showing how ribosomal RNA genes, which are clustered near the tips of five different human chromosomes (13, 14, 15, 21, and 22), come together to form the nucleolus, which is a biochemical subcompartment that consists of a dynamic assembly of many macromolecules. These components include ribosomal RNAs (rRNAs) and special proteins, in addition to the indicated DNAs.  Nucleosomes are the basic units of eukaryotic chromosome structure Nucleosomes can be seen in the electron microscope. (A) Chromatin isolated directly from an interphase nucleus can appear in the electron microscope as a chromatin fiber composed of packed nucleosomes. (B) Another electron micrograph shows a length of a chromatin fiber that has been experimentally unpacked, or decondensed after isolation to show the “beads-on-a-string” appearance of the nucleosomes.  Nucleosomes contain DNA wrapped around a protein core of eight histone molecules In a test tube, the nucleosome core particle can be released from chromatin by digestion of the linker DNA with a nuclease, which cleaves the exposed linker DNA but not the DNA wound tightly around the nucleosome core. When the DNA around each isolated nucleosome core particle is released, its length is found to be 147 nucleotide pairs; this DNA wraps around the histone octamer that forms the nucleosome core nearly twice.  Histone H1 provides additional packaging of nucleosomes in the chromatin fiber This “linker” histone binds to DNA, altering the DNA's path as it exits the nucleosome. In this way, histone H1 helps make the regions of chromatin that it associates with more compact.  SMC ring complexes use the energy of ATP hydrolysis to form chromatin loops Structural maintenance of chromosomes (SMC) ring complexes: motors that hydrolyze ATP to compact chromatin by forming loops 1. Cohesins: organize interphase chromatin 2. Condensins: compact interphase chromatin into mitotic chromosomes before cell division Although the detailed mechanism is still uncertain, the SMC complex works like a protein machine that uses energy supplied by ATP hydrolysis to perform this task. In one proposed mechanism, called the “inchworm” model, an SMC ring complex encircles and attaches to a DNA double helix. After hydrolyzing two molecules of ATP, the complex swings opens, widening its “grip” on the DNA. The subsequent release of ADP returns the complex to its original configuration, which brings its “feet” back together a little further along the DNA from where it started. Because the hinge at the top of the complex remains attached to one part of the DNA, this inching movement pushes the DNA at the foot of the complex out into a larger loop. Although the DNA shown here is “naked,” this chromatin fiber would be packaged into nucleosomes, which the SMC ring is large enough to accommodate. The same general mechanism applies to both the SMC ring complexes that organize interphase chromosomes (cohesins) and those that condense mitotic chromosomes (the condensins, to be discussed next).  Sequence-specific clamp proteins regulate the size of chromatin loops. (A) Cohesins will enlarge chromatin loops until they are stopped by clamp proteins that are bound to specific sequences of DNA. These clamp proteins then interact with one another, drawing together the DNA at the base of each loop. (B) Multiple cohesins operate to divide interphase chromosomes into an extended series of chromatin loops.  Condensins form loops within loops, folding a mitotic chromosome into a more compact configuration When cells enter mitosis, most of the cohesins (green) that organized the interphase chromosome are replaced by condensins. Mammalian cells have two condensins: condensin II (blue) forms the initial large chromatin loops and condensin I (purple) then forms a second set of loops inside them. This loops-within-loops organization, combined with the ever-tighter winding of these loops around the chromosome’s central axis, generates the compact structure of the mitotic chromosome.  DNA packing occurs at multiple levels in chromosomes Cohesins Condensins This schematic drawing shows the mechanisms thought to give rise to the highly condensed mitotic chromosome. Both histone H1 and a set of specialized non-histone chromosomal proteins help drive these condensations, including chromosome loop-forming clamp proteins and the SMC ring complexes, cohesin and condensin.  ATP-dependent chromatin-remodeling complexes locally reposition the DNA wrapped around nucleosomes Chromatic remodeling complexes Use ATP hydrolysis to shift positioning of nucleosomes on DNA Can expose or hide DNA sequences to regulate access of other DNA-binding proteins (A) The complexes use energy from ATP hydrolysis to pull on nucleosomal DNA, loosening its grip around the histone octamer. In this way, the enzyme can expose or hide a sequence of DNA, controlling its availability to other DNA-binding proteins. The blue stripes have been added to show how the DNA shifts its position. Many cycles of ATP hydrolysis are required to produce such a shift. (B) The structure of an ATP-dependent chromatin-remodeling complex, showing how the enzyme (green) cradles a nucleosome core particle, including a histone octamer (red, orange, blue, and dark green) and the DNA wrapped around it (gray). This large complex, purified from yeast, contains multiple subunits, including an ATP-driven motor (purple).  The pattern of modification of histone tails can determine how the cell handles a stretch of chromatin Histone tails are post-translationally modified in various patterns to regulate chromatin called the “histone code” Tails can be methylated, acetylated, and phosphorylated to promote condensation or expansion of chromatin Modifications made by “writers”, removed by “erasers”, acted upon by “readers” (A) Schematic drawing showing the positions of the histone tails that extend from each nucleosome core particle. Each histone can be modified by the covalent attachment of a number of different chemical groups, mainly to the tails. The tail of histone H3, for example, can receive acetyl groups (Ac), methyl groups (M), or phosphate groups (P). The numbers denote the positions of the modified amino acids in the histone tail, with each amino acid designated by its one-letter code. Note that some amino acids, such as the lysine (K) at positions 9, 27, and 36 (red), can be modified by acetylation or methylation (but not by both at once). Lysines, in addition, can be modified with either one, two, or three methyl groups; trimethylation, for example, is shown in (B). Note that histone H3 contains 135 amino acids, most of which are in its globular portion (represented by the wedge); most modifications occur on the N-terminal tail, of which 36 residues are shown. (B) Different combinations of histone tail modifications can confer a specific meaning on the stretch of chromatin on which they occur, as indicated. Although 100 or so histone modifications have been cataloged, only a few have been linked definitively with a particular functional outcome in terms of chromatin structure or gene expression.  The structure of chromatin varies along a single interphase chromosome Heterochromatin: highly condensed interphase chromatin containing “silent” genes that are not expressed Facultative: temporarily condensed; constitutive: permanently condensed (such as telomeres and centromeres) Euchromatin: less condensed interphase chromatin associated with genes that are being actively expressed As schematically indicated by the path of the DNA molecule (represented by the central black line) and the different arbitrarily assigned colors, heterochromatin and euchromatin each represent a set of different chromatin structures with different degrees of condensation. The constitutive heterochromatin in the centromere and telomeres remains permanently condensed, whereas the facultative heterochromatin has been condensed in only temporary manner. Although heterochromatin is more condensed than euchromatin, there are many stretches of euchromatin in which the resident genes are quiescent; these segments of “inactive” euchromatin (light green) are less extended than the “active” euchromatin containing genes that are expressed (dark green).  Heterochromatin-specific histone modifications allow heterochromatin to form and to spread Histone modifications can attract “reader–writer” protein complexes containing histone-modifying enzymes that identify and reproduce the same histone modifications on neighboring nucleosomes. In this manner, heterochromatin can spread until it encounters a barrier DNA sequence that blocks further propagation into regions of euchromatin. As also shown, histones bearing heterochromatin-specific modifications also recruit additional heterochromatin-specific proteins that drive chromatin condensation. This process can be reversed by histone-modifying enzyme complexes that recognize and remove heterochromatin-specific marks (not shown).  X chromosome inactivation silences gene on an entire chromosome One of the two X chromosomes is inactivated in the cells of mammalian females by heterochromatin formation. (A) Each female cell contains two X chromosomes, one from the mother (Xm) and one from the father (Xp). At an early stage in embryonic development, one of these two chromosomes becomes condensed into heterochromatin in each cell, apparently at random. At each cell division, the same X chromosome becomes condensed (and inactivated) in all the descendants of that original cell. Thus, all mammalian females end up as mixtures (mosaics) of cells bearing either inactivated maternal or inactivated paternal X chromosomes. In most of their tissues and organs, about half the cells will be of one type, and the rest will be of the other. (B) In the nucleus of a female cell, the inactivated X chromosome can be seen as a small, discrete mass of chromatin called a Barr body. In these micrographs of the nuclei of human fibroblasts, the inactivated X chromosome in the female nucleus (bottom micrograph) has been visualized by use of an antibody that recognizes proteins associated with the Barr body.  The male nucleus (top) contains only a single X chromosome, which is not inactivated and thus not recognized by this antibody. Below the micrographs, a drawing shows the locations of both the active and the inactive X chromosomes in the female nucleus.  Example of X-inactivation: the tortoiseshell cat’s coat color One of the genes specifying fur color is on X chromosome One X carries black fur, and the other carries orange fur Skin cells in which the X chromosome carrying the gene for black fur is inactivated will produce orange fur; those in which the X chromosome carrying the gene for orange fur is inactivated will produce black fur The tortoiseshell cat’s coat color is largely dictated by X-inactivation patterns. In cats, one of the genes specifying coat color is located on the X chromosome. In female tortoiseshells (or “torties”), one X chromosome carries the form of the gene that specifies black fur, the other carries the form of the gene that specifies orange fur. Skin cells in which the X chromosome carrying the gene for black fur is inactivated will produce orange fur; those in which the X chromosome carrying the gene for orange fur is inactivated will produce black fur. The size of each patch will depend on the number of skin cells that have descended from an embryonic cell in which one or other of the X chromosomes was randomly inactivated during development (see Figure 5– 31). (bluecaterpillar/Depositphotos.)  Epigenetic inheritance: heterochromatin can be inherited after DNA replication Heterochromatin can be inherited after DNA replication. When a chromosome is duplicated, the H3 and H4 histone proteins associated with the parent DNA are directly passed to the daughter helices. Thus, each daughter inherits half of these parent histones and their covalent modifications. Some of these modified histones attract “reader–writer” enzyme complexes that recognize the specific modification and spread it to nearby nucleosomes. This reestablishes the parental pattern of histone modifications and, hence the parental chromatin structure. Because chromatin structure regulates gene expression, this form of epigenetic inheritance helps to provide daughter cells with the instructions they need to retain the identity of their parent cell.