Chromosome, Eukaryotic

Basic Organization

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.

Higher-Order Organization

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.

Cytological Features

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.

Polytene Chromosomes

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.

Cynthia Guidi

and Anthony N. Imbalzano

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