The deoxyribonucleic acid (DNA) of eukaryotic cells carries the blueprint for the biosynthesis of cellular proteins and the control of cellular assembly and regulation. If all the DNA in a single human cell were stretched out straight and the strands representing all the chromosomes laid end-to-end, they would extend for well over 1 meter (3 feet). This meter of DNA must fit into a nucleus whose diameter is on the order of 10 microns (10–5 meter)! The dual problem of how to store this large amount of genetic information but also to keep it accessible for use and for faithful maintenance, copying, and distribution to daughter cells during cell division, is solved by using proteins to package the DNA into chromosomes.
During the cell cycle , the cell grows (during G1 phase), replicates its DNA (during S phase), prepares for cell division (during G2 phase), and divides by mitosis (during M phase). During M phase, each chromosome is duplicated, and each replica remains attached to its original at the centromere portion of the chromosome. The two identical strands, called chromatids , wind up and become visible under the microscope at the beginning of mitosis. During the portion of mitosis known as metaphase , spindle fibers (which attach to the centromeres) jostle the chromatid pairs to the middle of the cell. The two chromatids are then pulled apart and segregated into different daughter cells, ensuring that each new cell has identical genetic information. The cells then enter G1 phase again. The combination of G1, S, and G2 is known as interphase. During interphase, the genes carried on the chromosomes are transcribed , to form proteins needed by the cell.
Various proteins act to stabilize DNA in interphase, while additional proteins are required to condense the chromosomes over a thousandfold to form the compact chromosomes required for mitosis and cell division. The sections that follow summarize key concepts concerning the structure of eukaryotic chromosomes.
Histones and Nucleosomes
Nearly all of the DNA in eukaryotic cells is complexed with a set of small basic proteins called histones . (As its name suggests, DNA is acidic, and is attracted to the basic histones.) The complexes form a repeating unit, the nucleosome, which consists of an octomeric disc of histones with about two turns of DNA wrapped around the outside. Thus, chromosomal DNA is organized as a string of nucleosome beads with a small amount of DNA connecting each bead. This first level of organization helps to compact the DNA so it can fit into the nucleus while still affording the necessary flexibility to fold the chromosome further; for example, in the condensation of chromosomes at metaphase.
The structure of the nucleosome is known at the atomic level through X-ray crystallography . The histone proteins interact extensively with one another to form the compact central disc of the nucleosomes, while specific amino acids have been identified that hold the DNA tightly onto the nucleosome surface. However, about fifteen to twenty-five amino acids at the end of each histone extend outside the compact limits of the central protein core. These tails are invisible in the X-ray structure of the nucleosome, indicating that they are relatively unstructured. (X-ray crystallography can only utilize structures with a high degree of order.) This indicates they can accommodate dynamic interactions with DNA or with adjacent nucleosomes in living chromosomes.
The sequence information encoded in DNA must be accessible to ribonucleic (RNA) polymerases in order to be useful as a template for transcription . Since the binding of DNA by histones interferes with this access, cells have evolved specific mechanism to destabilize nucleosomes in chromosome regions that must be transcribed. While the details of this important process are still being deciphered, it is clear that there are enzymes in eukaryotic nuclei that can modify nucleosome structure or the structure of individual histones to loosen the histone-DNA contacts, thereby making the DNA available for transcription.
One class of enzyme believed to modify nucleosomes for transcription is the histone acetyltransferases, which catalyze acetylation of specific lysines in the N-terminal tails of histones. Acetylation of lysines reduces the overall positive charge of the histone protein; since DNA has a net negative charge, histone acetylation may reduce the electrostatic forces holding the DNA on the nucleosome. This is thought to make the DNA more accessible to other DNA-binding proteins such as RNA polymerase .
In addition, some transcription regulatory proteins bind more easily to their DNA target sites if the nucleosomes associated with those sites are acetylated. A critical part of the transcription activation mechanism in eukaryotic cells appears to be the specific recruitment of nucleosome remodeling enzymes, such as histone acetyltransferases to promoters , thus allowing those promoters to be used more efficiently by RNA polymerase. Histone acetylation, therefore, can increase the transcription rate for a gene. Conversely, cells also possess histone deactylases. Histone deacetylases may be specifically recruited to shut off genes when they are no longer required.
Another type of histone modification is addition of a phosphate group called phosphorylation . Phosphorylation typically causes significant changes in protein structure and activity. Increased histone phosphorylation is correlated with chromosome condensation at the onset of mitosis. The mechanism by which phosphorylation promotes condensation is unclear, but may involve nucleosome-nucleosome interactions, or the binding of nonhistone proteins to nucleosomal DNA as part of the folding of chromosomes for metaphase.
30 Nanometer Fiber
The nucleosomal organization of DNA in chromosomes cannot fully account for the degree of compaction necessary to fit the genome into the compact nucleus. The nature of these additional levels of DNA folding is controversial, but is believed to include the coiling of nucleosome arrays to form a solenoidal structure. Such solenoids have been visualized in electron micrographs of eukaryotic chromosomes as fibers of 30 nanometers (a nanometer equals one billionth of a meter) in diameter, in contrast to the 10-nanometer diameter of the nucleosome particle itself. In somatic nuclei, the 30-nanometer fiber appears to be stabilized by a specific histone, histone H1, which interacts with the DNA-linking adjacent nucleosomes.
Domains and Higher Order Structures
Early electron micrograph images of eukaryotic metaphase chromosomes gave the impression of looped fibers extending out from the central axis of each chromatid. Subsequent analysis by microscopic and biochemical techniques suggests that stretches of chromosome approximately forty thousand to eighty thousand nucleotide pairs long may be anchored to a nuclear scaffold or matrix . These points of anchorage may serve to organize or spatially restrict chromosomes during interphase. These same anchor points may coalesce at metaphase to condense chromosomes for mitotic segregation.
An average human chromosome contains approximately 240 million base pairs.
Chromosomes exist to hold genes, of course, and some structural features of the chromosome may serve to separate genes from one another to help regulate transcription. Gene transcription in higher eukaryotes is controlled by regulatory elements that, in some cases, are located hundreds of thousands of nucleotides away from their target promoters. How can such elements be prevented from activating other nearby promoters? Experiments suggest that there are DNA sequences that act as boundaries or barriers to prevent the distant regulatory elements from one gene from contacting the promoters of genes located elsewhere on the same chromosome. In some cases, these genetic domain borders may be equivalent to the nuclear scaffold/matrix anchorage points, but in other cases these activities appear separable.
Telomeres, Telomerase, and Cancer
In his studies of chromosome structure, geneticist Herman Muller recognized that the natural ends of chromosomes were peculiar in that they could not be placed at internal sites in chromosomes, and that if they were detached (by breakage with ionizing radiation ), the resulting chromosome behaved abnormally. He recognized the special properties of chromosome ends by giving them a special name: "telomeres." Scientists now know that the ends of chromosomes have a unique structure and are maintained by a unique mechanism.
The chromosomes of eukaryotic cells are linear DNA molecules. Because of this fact, and because of the mechanics of normal DNA replication by DNA-dependent DNA polymerases, a small amount of DNA at each end of every chromosome fails to be replicated with every cell cycle in somatic cells. If this loss occurred in the germ line as well, all eukaryotes would become extinct after a few generations, as important genes located near the chromosome ends would eventually be lost by the gradual chipping away at the ends. The major way that living cells offset this loss is by adding extra DNA onto one strand using a special enzyme for this purpose called "telomerase." Telomerase is a ribonucleoprotein complex, consisting of an RNA-dependent DNA polymerase (also known as a reverse transcriptase ) and an RNA molecule that serves as a template for DNA synthesis, giving rise to the characteristic repeated DNA sequence of most eukaryotic telomeres. (The fruit fly Drosophila is a notable exception to this; it uses transposable elements to maintain its telomeres.)
Telomerase activity in metazoans is found primarily in germ cells and at low levels in a few somatic tissues (stem cells that give rise to blood and skin cells that have to be replenished constantly throughout adult life). Normal animal somatic cells that are cultured in vitro usually lack telomerase activity. Such cells typically can divide only a finite number of times before they stop proliferating, go into a quiescent state, and eventually die, a process called senescence. Sencescence in cultured cells is correlated with loss of telomeric repeats. In general, cancer cells escape senescence and often can proliferate indefinitely in culture; this phenomenon, called immortalization, is accompanied by the activation of telomerase activity. Although cancer cells are often found to have unusually short telomeres, the length of their telomeres remains stable as the cells continue to proliferate. It is believed that telomerase activation in cancer is essential to continuous tumor growth and metastasis . Since most somatic cells have low or undetectable telomerase activity, drugs that specifically inactivate telomerase activity should be potent anticancer drugs with minimal side effects on healthy normal tissue.
Condensation and Decondensation
While chromosomes undergo cycles of condensation and decondensation with entry into and exit from mitosis during the cell cycle, some regions of chromosomes remain condensed throughout most of interphase. This chronically condensed material in the nuclei of all eukaryotic cells was recognized by German cytogeneticist Emil Heitz, who named it "heterochromatin" (in contrast with the "euchromatin," or "true chromatin"), which disperses with the onset of interphase. The regions surrounding most eukaryotic centromeres is composed of heterochromatin.
In mealybugs, the entire paternal genome set is inactivated by heterochromatinization early in development, and therefore mealybugs express only maternally derived genes.
Heterochromatin is distinguished from euchromatin by other properties. It replicates late in S phase while euchromatin replicates early in S, and it has the ability to silence euchromatic genes. Biochemical analysis shows that the DNA in heterochromatin is less accessible to a variety of DNA-binding proteins, suggesting that heterochromatin condensation inactivates regions of chromosomes by interfering with the accessibility of DNA for transcription. In mammalian females, one X chromosome is inactivated by heterochromatinization. This is thought to ensure that both males (who have only one X) and females (who have two) have equal "doses" of the many genes carried on the X chromosome.
Classes of DNA
The chromosomes of higher eukaryotes contain classes of DNA sequences that differ in the number of times they are presented in the genome. Much of the DNA in higher eukaryotes is unique, in the sense that the exact linear sequence of nucleotides is found only once per haploid chromosome complement. But some DNA sequences are found in a few dozen or a few hundred identical or nearly identical copies in each haploid chromosome set. These are considered "moderately repetitive" DNA sequences, and in most higher eukaryotes include the genes encoding the histones and the ribosomal RNA (rDNA), as well as certain classes of transposable elements. In the case of the repeated histone and rDNA, having many copies of these genes may be important at certain stages of development to allow biosynthesis of large amounts of histone proteins (during S phase) and ribosomal RNA (during ribosomal synthesis) in a short period of time.
Extra X chromosomes, as in XXY males or XXX females, are also condensed, to leave only one active X chromosome.
The third broad class of DNA found in higher eukaryotic chromosomes is represented in many thousands of copies, and is thus termed "highly repetitive." Because of the relative abundance and sequence homogeneity of highly repetitive DNA sequences, they were initially isolated from fragmented eukaryotic DNA as "sattelites" easily separated from the main mass of DNA. This satellite DNA includes tandem arrays—many copies, one right after another—of a 171-nucleotide pair repeat called "alphoid satellite." Alphoid satellite DNA is found in tandem arrays of thousands of copies in the centromeres of all human chromosomes. The alphoid repeats are sufficient to confer centromeric properties on artificial human chromosomes. (The centromere region forms the "pinched waist" so characteristic of metaphase chromosomes, and is the site to which the spindle fibers attach to separate daughter chromatids in mitosis.
The function of other types of highly repetitive sequence DNA is unknown; indeed, some repetitive DNA sequences are thought to be "junk DNA," present in chromosomes simply because there is no evolutionarily efficient way to eliminate it. Approximately 500,000 copies of a 300-nucleotide-pair sequence called an "Alu sequence" are found in the human genome. Unlike the alphoid satellite, Alu sequences are interspersed throughout all human chromosomes. Alu sequences are homologous to portions of the 7SL RNA, a structural component of the signal recognition particle that targets ribosomes to the endoplasmic reticulum . Alu sequences are probably relics of reverse transcription of this RNA into 7SL DNA, which then recombined randomly into chromosomes. Such dispersed repeated DNA sequences are potential sites for homologous recombination , not only between noncorresponding positions on the same chromosome or on different chromosomes. Indeed, recombination between Alu elements is probably responsible for some deletion or rearrangement of mutations leading to inherited human diseases, since Alu sequences are often found at deletion/rearrangement breakpoints.
Hereditary nonpolyposis colon cancer is associated with defects in mismatch repair.
Throughout all chromosomes of all living organisms, short, simple sequence repeats may be found. For example, short stretches of guanosine-cytosine base pairs , alternating adenosine-thymidine and cytosine-guanosine, occur randomly, both within and outside of protein-coding sequences, and are sometimes referred to as "microsatellite repeats." In such regions, there is a higher tendency for the DNA polymerase to make errors by skipping a nucleotide or adding a couple of nucleotides. Such errors create sites of mismatched bases, which could lead to mutation—and cancer—if they are inherited by daughter cells after cell division. Most living cells have a way of detecting and correcting such mismatches shortly after they occur, using a mechanism termed "mismatch repair." Patients that lack one of the components of the mismatch repair machinery have a much higher chance of being victims of certain types of cancers.
Numbers and sizes of chromosomes vary widely in eukaryotes, and neither correlates with genome size. The classification of chromosomes within a given species was made possible initially by the used of stains that revealed variation in the DNA sequence composition along the length of the chromosome, resulting in a banded staining pattern characteristic for each chromosome. Using the criteria of overall chromosome length, relative centromere position and banding pattern, chromosomes of any species can be identified as a characteristic ordered set called a karyotype. With advent of molecular hybridization and extensive molecular cloning of unique-sequence DNAs, DNA sets representing sequences unique to individual chromosomes have been identified. By coupling the cloned DNA to fluorescent dyes and hybridizing the fluorescently labeled DNA directly to chromosomal preparations or whole cells, fluorescent in situ hybridization
Rett syndrome is a rare genetic disorder resulting from defects in a methylcytosine-binding protein, MeCP2. Rett syndrome affects girls, and causes slowed development, mutism, and seizures.
(FISH) enables rapid, efficient, and reliable identification of whole chromosomes or chromosome fragments. FISH has found widespread clinical application in the identification of chromosome rearrangements underlying inherited disease and many tumors.
Cytosine Methylation and Gene Regulation
When cellular DNA is first replicated, it consists of four nucleotide subunits: deoxyadenosine, deoxycytidine, deoxyguanosine, and thymidine. Following DNA replication, though, chemical modifications can occur to DNA. One of the most commonly encountered modifications found in the DNA of mammalian cells is the methylation of cytidine at carbon number 5 of the cytosine base. In human cells, about 3 to 5 percent of the cytosines are so methylated. The distribution of methylated sites is not uniform, but occurs only at cytosine residues that precede a guanosine (so-called CpG motifs, where the "p" symbolizes the intervening phosphate in the sugar-phosphate DNA backbone). Clusters of CpG dinucleotides—called CpG islands—preferentially occur near the promoters of many mammalian genes. When the cytosines in such islands are extensively methylated, the gene associated with that island is usually found to be transcriptionally silent. Thus, cytosine methylation is inversely correlated with gene expression . The mechanism of methylation-dependent silencing involves proteins that specifically recognize and bind to methylated DNA
Cytosine methylation is also found in plants, where it is also inversely correlated with gene activity. Interestingly, many fungi and insects have no detectable DNA methylation at all, yet they seem to be able to regulate their genes adequately. One theory is that DNA methylation arose in evolution as a secondary mechanism to ensure faithful gene silencing in organisms that undergo many cell divisions in development between fertilization and adulthood. It may also have evolved to inactivate certain types of viruses.
see also Cell Cycle; Chromosome Aberrations; Control of Gene Expression; DNA; Gene; Nucleotides; Oncogenes and Cancer Cells; Sex Chromosomes; Transposon
Joel C. Eissenberg
Greider, Carol W., and Elizabeth H. Blackburn. "Telomeres, Telomerase and Cancer." Scientific American 274, no. 2 (1996): 92–97.
Grunstein, Michael. "Histones as Regulators of Genes." Scientific American 267, no.4 (1992): 68–74B.
Moxon, E. Richard, and Christopher Wills. "DNA Microsatellites: Agents of Evolution?" Scientific American 280, no.1 (1999): 94–99.
Living organisms are divided into two broad categories based upon certain attributes of cell structure. The first category, the prokaryotes, includes bacteria and blue-green algae. Eukaryotes include most other living organisms. One of the most important features distinguishing eukaryotes from prokaryotes is the chromosomal arrangement of genetic information in the cells. Eukaryotes enclose their genetic material in a specialized compartment called the nucleus. Prokaryotes lack nuclei.
In 1883, Wilhelm Roux proposed that the filaments observed when cell nuclei were stained with basic dyes were the bearers of the hereditary factors. Heinrich Wilhelm Waldeyer later coined the word chromosome ("colored body") for these filaments. The eukaryotic chromosome now is defined as a discrete unit of the genome, visible only during cell division, that contains genes arranged in a linear sequence. Eukaryotic organisms contain much more genetic information than prokaryotes. For example, the eukaryotic organism Saccharomyces cerevisiae (baker's yeast) contains 3.5 times more DNA in its haploid state than the prokaryotic Escherichia coli, while higher vertebrate cells contain more than 1,000 times the DNA.
The basic component of the eukaryotic chromosome is its DNA, which contains all of the genetic material responsible for encoding a particular organism. Genes are arranged in a linear array on the chromosome. A major distinction between eukaryotic and prokaryotic chromosomes is that eukaryotic chromosomes contain vast amounts of DNA between the genes. The function of most of this "extra" DNA is unknown. It contains repetitive sequences, functionless gene copies called pseudogenes, transposible elements, and other types of DNA.
Eukaryotic genes may be dispersed randomly throughout the chromosome or they may be specifically organized. A gene family is a set of genes that originated from the duplication and subsequent variation of a common, ancestral gene. Members of a gene family may be clustered on the same chromosome, as in the case of the globin genes. Gene duplication events also have resulted in gene clusters in which related or identical genes are arranged in tandem. Examples of gene clusters include the genes for rRNA and histone proteins.
The DNA of a eukaryotic cell must be constrained within the confines of the nucleus. In human cells, six billion base pairs are contained on the forty-six chromosomes of double-stranded DNA. This DNA has a total length of 1.8 meters, yet it must fit into a nucleus with an average diameter of 6 micrometers . This feat is accomplished in part by the packaging of DNA into chromatin, a condensed complex of DNA, histones, and nonhistone proteins.
The basic unit of chromatin is the nucleosome. The nucleosome is composed of approximately 146 base pairs of DNA wrapped in 1.8 helical turns around an eight-unit structure called a histone protein octamer. This histone octamer consists of two copies each of the histones H2A, H2B, H3, and H4. Nucleosomes form arrays along the DNA. The space in between individual nucleosomes is referred to as linker DNA, and can range in length from 8 to 114 base pairs, with 55 base pairs being the average. Linker DNA interacts with the linker histone, called H1, and there are equal numbers of nucleosomes and H1 histone molecules in the chromatin. A nucleosome particle bound to a single molecule of H1 is termed a chromatosome.
Higher-order chromatin structure can be visualized microscopically as fibers 10 and 30 nanometers in diameter. The 10-nanometer fiber can be observed under conditions of low ionic strength. This fiber resembles beads on a string, and is in fact a string of nucleosomes. The structure does not require the presence of histone H1. It is unclear if the 10-nanometer fiber exists in vivo or if it is just an artifact of chromatin unfolding during extraction in vitro .
Cellular chromatin usually exists as a 30-nanometer fiber. It can be seen under conditions of higher ionic strength. The presence of the linker histone H1 is required for the formation of this fiber, as it helps promote compaction and condensation. The 30-nanometer fiber is formed into a coil, but its exact structure has not been determined.
During cell division, chromosomes condense to an even greater extent. The mechanism by which the 30-nanometer fibers are packed into the highly condensed, organized structure of the mitotic chromosome is unclear. The compaction of chromosomes during cell division is accomplished in part by the organization of chromatin into large, looped structures that are attached at their bases to a protein scaffold. This scaffold remains intact even if the DNA is experimentally removed. It is possible that this scaffold is responsible for maintaining the shape of the chromosomes.
Heterochromatin versus Euchromatin
Chromatin can be divided into two regions, euchromatin and heterochromatin, based on its state of condensation, that is, based on how tightly its constituent elements are packed together. Most of the cellular chromatin is euchromatin, which has a relatively dispersed appearance in the nucleus. It condenses significantly only during mitosis. Genes within euchromatin can be transcriptionally active or repressed at a given point in time.
Heterochromatin, on the other hand, is condensed in interphase , usually does not contain genes that are being expressed, and is among the last portions of the genome to be replicated prior to cell division. Heterochromatin frequently is localized at the periphery of the nucleus. It can be subdivided into constitutive and facultative heterochromatin. Constitutive heterochromatin is always inactive. It is often found adjacent to centromeres and telomeres. Facultative heterochromatin refers to DNA sequences that are specifically inactivated as the result of development or a regulatory event. One example of facultative heterochromatin is the mammalian X chromosome. The single X chromosome present in male cells is active. However, in female cells, one of the two copies present is directly and specifically inactivated.
While the interphase chromatin appears to be a tangled mass within the nucleus, the mitotic chromosome appears as an organized structure with many prominent features. These features include structures known as the centromere and the telomere .
The centromere is the region of the chromosome to which the spindle apparatus attaches during mitosis and meiosis. The spindle apparatus is the network of fibers along which the chromosomes move during cell division. It also contains the site at which sister chromatids are attached prior to their separation during the stage of cell division known as anaphase. The centromere is responsible for the movement of the chromosome. During mitosis and meiosis, the centromere is pulled by the spindle fibers toward the opposite ends of the dividing cell (poles), as the attached chromosome is dragged behind. The centromere is essential for segregation.
The telomere is a structure that occurs at the end of linear eukaryotic chromosomes and that confers stability. The first telomere to be sequenced was from the organism Tetrahymena thermophila, a type of single-cell eukaryotic organism, in 1978 by Elizabeth Blackburn and Joseph Gall. This telomere contains an AACCCC nucleotide sequence that is repeated thirty to seventy times. The sequences of telomeres from other species show the same pattern: a tandem array of a short nucleotide sequence, one DNA strand Grich and the other DNA strand C-rich. Telomeres are synthesized by an enzyme called telomerase, which adds telomeric sequences back onto chromosome ends, one base at a time.
Banding techniques allow every mitotic chromosome, as well as regions within individual chromosomes, to be distinguished. The first method used, known as Q-banding, uses flourescent derivatives of quinacrine, which for unknown reasons bind preferentially to some regions of chromosomes. When viewed under ultraviolet light, chromosomes appear with bright bands, corresponding to euchromatin, and dark bands, corresponding to heterochromatin. This banding method is useful in identifying some polymorphisms, which are gene variations (alleles ) within the population of genomes. It is also useful for identifying the Y chromosome.
Later, another banding technique was developed, called G-banding. This technique employs a modified Giemsa stain, which is a dye that specifically binds to DNA. As in Q-banding, a series of identifiable light and dark bands is generated on the chromosome. Euchromatin stains lightly, while most heterochromatin stains darkly.
These banding methods have made it possible to diagrammatically represent each human chromosome, using designated nomenclature for specific chromosomal regions. These techniques have shown that the mitotic chromosome can be divided into a short arm, designated p, and a long arm, q. Each arm is then divided into one to three regions by landmarks, such as the ends of the arm, the centromere, and certain prominent bands. Regions are spaces between adjacent landmarks and are numbered consecutively within each region. These features allow genes to be designated to specific regions on the mitotic chromosome. For example, a gene at band 4q14 is localized to chromosome 4, the long arm, region 1, band 4.
Due to the compact nature of chromatin in eukaryotic cells, it is virtually impossible to visualize gene expression in vivo. However, there are certain unusual situations in which gene expression can be seen. Such is the case with the polytene chromosomes, which are exceptionally large in comparison to other types of chromosomes.
The salivary glands of Drosophila melanogaster (fruit fly) larvae contain greatly enlarged chromosomes. These are polytene chromosomes, and they result from multiple rounds of replication of a diploid pair of chromosomes joined in parallel. The replicated chromosomes remain attached to one another. Each pair of chromosomes can replicate up to nine times; thus, the resultant polytene chromosome can contain up to 1,024 (29) strands of DNA.
The vast majority (95%) of DNA in the polytene chromosomes is concentrated in chromosomal bands, called chromomeres, which are microscopically visualized through staining. These chromomeres form a pattern that is characteristic for each Drosophila strain. Drosophila polytene chromosomes display roughly 5,000 bands. Since the total number of genes in Drosophila appears to be greater than the number of bands that can be visualized, it is likely that there are multiple genes located within a given band.
The banding pattern of the polytene chromosomes provides a cytological map, or diagrammatic representation, of the physical location of genes at specific sites in the cell. The positions of individual genes can be determined using a technique called in situ hybridization. First, the DNA of an immobilized chromosome preparation is made single-stranded (denatured). A radioactively labeled probe, generally a small piece of DNA corresponding to the gene of interest, is then mixed with the denatured DNA under conditions that permit the radiolabeled DNA to bind to its complementary DNA strand on the immobilized chromosome preparation. Finally, the chromosomal binding site is determined by way of a procedure called autoradiography, which is used to make the radioactive probe visible on photographic film.
With autoradiography, the sites of gene expression can be visualized along the polytene chromosomes. As DNA decondenses into a more open state, it forms a distinctive swelling known as a chromosomal puff. These puffs are active sites of transcription, or RNA synthesis. Throughout development, they alternately expand and contract, as specific genes are activated or repressed.
Chromosome Organization, Replication, and Transcription
The compact nature of chromatin structure presents a barrier to processes that require access to DNA, such as replication and transcription. It seems likely that the separation of parental DNA strands during replication must disrupt higher-order chromatin structure to at least some degree. In the Drosophila polytene chromosomes, this disruption can be seen, at least in part, as puffs at sites of DNA replication. It is unclear, however, what happens to the nucleosomes during this process. If the nucleosomes are removed during replication, they are quickly reassembled, for there is no time period during which microscopic analysis shows the DNA to be free of nucleosomes.
Transcriptional activation of eukaryotic genes requires that the machinery responsible for synthesizing the RNA gain access to the regulatory regions of the DNA that control gene expression. This again necessitates some decondensation of the chromatin structure. Decondensation can be facilitated by protein complexes known as chromatin-remodeling enzymes . Chromatin-remodeling enzymes alter the structure of chromatin in such a way that regulatory factors can gain access to the DNA. These enzymes are divided into two groups, those that chemically modify histones and those that use the energy derived from ATP hydrolysis to alter histone-DNA linkages.
Chemical Modification of Chromatin Structure
The best-characterized of the enzymes capable of chemically modifying histones to open chromatin structure are the histone acetyltransferases, or HATs. These enzymes add acetyl groups to lysine residues within the amino termini (also known as the tails) of H3 and H4 histones. This adds a negative charge to the histone tails. The negative charge is believed to cause them to push away from the DNA backbone, resulting in a somewhat less condensed chromatin structure. Hyperacetylation of histones in the promoter regions of genes is associated with active, ongoing gene expression, while histone hypoacetylation is associated with genes that are transcriptionally silent.
The association between histone acetyltransferases and gene activation was first suggested when it was found that the Tetrahymena thermophila HAT p55 was structurally similar to the yeast protein GCN5. GCN5 previously had been shown to be involved in gene activation. Later it was discovered that GCN5-regulated genes are hyperacetylated when active, and that certain mutations in GCN5 that affect its ability to activate target genes result in diminished levels of acetylation of these regions. Histone acetyltransferases also are found in mammalian cells. It is believed that the HATs are recruited to specific genes by specific transcriptional activators.
The activity of the histone acetyltransferases is opposed by histone deacetylases. These enzymes remove acetyl groups from the histone tails, resulting in the repression of gene activation. As with the HATs, these enzymes are believed to be recruited to chromatin by repressor proteins to aid in the inactivation of specific genes.
Transcriptional activation also can be repressed by the methylation of specific cytosine residues found in some genes. It is unclear how methylation results in transcriptional repression, but it is known to cause the inhibition of specific transcriptional activators and to initiate the recruitment of specific repressors that bind methylated DNA. At least one methylated DNA-binding repressor can be isolated from cell extracts together with (and therefore appears to be associated with) histone deactylases, thereby providing a link between methylation and deacetylation and gene inactivation. Other enzymes chemically modify histones by adding or removing phosphate, ubiquitin, and other chemical groups.
ATP-Dependent Chromatin-Remodeling Complexes
The presence of enzymes that can alter the structure of chromatin was suggested by yeast genetic studies that identified a number of genes, called SWI and SNF genes. These genes are required for multiple transcriptional activation events. A key breakthrough came when it was discovered that yeast cells could compensate for a deficiency in these SWI and SNF gene products by altering their chromatin structure. This led to the hypothesis that SWI and SNF genes are involved in the regulation of chromatin structure. It is now known that SWI and SNF proteins form a large, multisubunit complex, termed SWI/SNF, that can hydrolyze ATP and use the energy thus generated to alter chromatin structure.
Similar proteins that can hydrolyze ATP are present throughout the eukaryotic kingdom, and these form related multiprotein enzymes that also possess chromatin-remodeling properties. The mechanism by which these complexes alter the chromatin structure is unclear, but it is likely that the enzymes break or loosen the linkage between histone and DNA in a manner that increases the mobility and flexibility of the DNA wrapped around the histone core. It is important to keep in mind that these enzymes can be involved both in gene-activation events, by facilitating the binding of transcriptional activators, and in gene-repression events, perhaps by facilitating the binding of a transcriptional repressor or by directly promoting compaction of the chromatin structure.
see also Cell Cycle; Cell, Eukaryotic; Chromosomal Banding; Chromosome, Prokaryotic; DNA; Evolution of Genes; Gene; Gene Expression: Overview of Control; In situ Hybridization; Meiosis; Mitosis; Mosaicism; Repetitive DNA Elements; Replication; Telomere; X Chromosome; Y Chromosome.
and Anthony N. Imbalzano
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