Chromosomes are typically the largest macromolecules in any cell, by a considerable margin. Why must chromosomes be so large? They contain the blueprints for an organism, and thus a great deal of information is contained within them. But the genes in each chromosome constitute only part of that information. The chromosomes themselves are macromolecular entities that must be synthesized, packaged, protected, and properly distributed to daughter cells at cell division. Significant segments of every chromosome are dedicated to these functions. All aspects of chromosome function are affected by the reality of chromosome size.

The chromosomes of cells and viruses come in several forms. Bacterial chromosomes are often circular (in the sense of an endless loop rather than a perfect circle). Most eukaryotic chromosomes are linear. In viruses we find additional variations, including both single-stranded and double-stranded forms, as well as RNA genomes. Each type of chromosome structure imposes a unique set of demands on the mechanisms for replicating and transmitting the genome from one generation to the next.

Genes provide the information to specify all the RNAs and proteins produced in a given cell, but other DNA sequences in the genome are dedicated to the maintenance of the chromosome itself: initiation and termination of replication, segregation during cell division, and, where necessary, protection and maintenance of the chromosome ends. In bacteria, an origin of replication provides a start site for chromosomal replication (see Figure 8-1). Specialized replication-termination regions also exist in most known bacterial species. Within or near these regions, additional sequences serve as binding sites for proteins that ensure the faithful segregation of replicated chromosomes to daughter cells. Eukaryotic chromosomes, too, contain sequences that are critical to chromosome maintenance. Unlike bacteria, eukaryotic chromosomes often have many replication origins. (The structure and function of replication origins are discussed in Chapter 11.) Eukaryotic chromosomes also have specialized DNA sequences called centromeres and telomeres.

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The centromere is a segment of each eukaryotic chromosome that functions during cell division as an attachment point for proteins that link the chromosome to the mitotic spindle at metaphase (Figure 9-1). This attachment is essential for the equal and orderly distribution of chromosome sets to daughter cells. (See Chapter 2 for a review of the events of mitosis.) The centromeres of Saccharomyces cerevisiae have been isolated and studied. The sequences essential to centromere function are about 130 bp long and very rich in A = T base pairs. The centromere sequences of higher eukaryotes are much longer and, unlike those of yeast, generally contain regions of simple-sequence DNA consisting of thousands of tandem copies of one or a few 5 to 10 bp sequences. This DNA serves as a binding site for centromere-binding proteins, or cen proteins. The centromere is also the site of kinetochore assembly. Built up on each centromere, the kinetochore anchors the spindle fibers as chromosomes are segregated into daughter cells during mitosis. Centromeres thus play a key role in stable chromosome segregation during cell division.

Telomeres are sequences at the ends of eukaryotic chromosomes that add stability by protecting the ends from nucleases and providing unique mechanisms for the faithful replication of linear DNA molecules. DNA polymerases cannot synthesize DNA to the very ends of a linear chromosome (see Chapter 11). Solving the end-replication problem is one key function of telomeres, which are replicated by the enzyme telomerase. Telomeres end with repeated sequences of the form

5′-(T_xG_y)_n

3′-(A_xC_y)_n

where x and y are generally between 1 and 4 (Table 9-1) and the number of telomere repeats, n, is in the range of 20 to 100 for most single-celled eukaryotes and generally exceeds 1,500 in mammals. As in centromeres, the telomere repeats serve as binding sites for specialized proteins that are part of telomere function. These proteins package the telomeres and help maintain them in actively dividing cells (see Chapter 11).

Artificial chromosomes provide a means of better understanding the functional significance of many structural features of eukaryotic chromosomes. A reasonably stable artificial linear chromosome requires only three components: a centromere, a telomere at each end, and an appropriate number of replication origins. Yeast artificial chromosomes (YACs) have been developed as a research tool in biotechnology (see Figure 7-7). YACs have also been useful in confirming the critical functions of centromeres and telomeres. Building on this foundation, researchers have constructed human artificial chromosomes (HACs). HACs are reasonably stable when introduced into a human tissue culture cell line, if they include human centromere and telomere sequences in addition to active replication origins.

Continued development of HACs, particularly of their efficient introduction into human cells, may eventually provide new avenues for the treatment of genetic diseases. Most genetic diseases can be traced to an alteration in one or more particular genes that changes or eliminates their function. Efforts to directly remove such genes in human cells and replace them with normal, functional versions at the correct chromosomal locus have met with limited success. Nonetheless, there are ongoing efforts to improve this process of correcting disease-causing genetic errors in somatic cells, a process termed somatic gene therapy. The CRISPR/Cas9 system, described in Section 7.3, provides a promising new approach to this effort. A simpler and more traditional path for gene therapy is to introduce the functional genes into random locations on chromosomes through recombination mechanisms (see Chapters 13 and 14). However, this technique has a number of problems. The inserted gene can run afoul of regulatory mechanisms that suppress gene expression over large segments of a chromosome, effectively silencing any new gene that is inserted there. Random integration can also result in insertion into the coding sequence of another gene, inactivating that gene. If the inactivated gene has a role in the regulation of cell division, uncontrolled cell division and tumor development can result. The introduction of functional gene copies on stable HACs may eventually circumvent these problems. Success will depend on further clarifying the mechanisms by which chromosomes are stably maintained in cells, and on the development of more efficient procedures for introducing large DNAs into the nuclei of a large number of cells in a living human being.

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The observation that genomic DNAs are orders of magnitude longer than the cells or viruses that contain them applies to every class of organism and viral parasite. Lengths of double-stranded nucleic acids are often described in terms of contour length, or the length measured along the axis of the double-helical DNA. For a closed-loop DNA, contour length is the circumference the chromosome would have if it were laid out in a perfect circle. Lengths are more difficult to describe for a single-stranded nucleic acid, particularly when segments of that nucleic acid fold up into secondary structures. These lengths are sometimes approximated by assuming that the single strand is arrayed in the helical path that would be described by one strand of a double helix, then measuring along the resulting axis.

Given the magnitude of the one-dimensional length of a typical chromosome, how can it be accommodated within the three-dimensional volume of a viral particle, cell, or nucleus? The compaction mechanisms required for this are highly conserved across the spectrum of living systems. Compaction entails the coiling and structural organization of the chromosome resulting from the action of enzymes; the structural organization is maintained by DNA-binding proteins, including the histones of eukaryotic chromosomes (see Chapter 10), the DNA-binding proteins of bacteria, and the coat proteins of viral particles. We consider here the chromosomes of viruses and of each class of living organism.

Viruses Viruses are not free-living organisms; they are infectious parasites that use the resources of a host cell to carry out many of the processes they require to propagate. Many viral particles consist of no more than a genome (usually a single RNA or DNA molecule) surrounded by a protein coat.

Almost all plant viruses and some bacterial and animal viruses have RNA genomes, and they are quite small. For example, the genomes of mammalian retroviruses, such as HIV, have about 9,000 nucleotides, and the genome of the bacteriophage Qβ has 4,220 nucleotides. However, even these small nucleic acids have total lengths of about 3 and 1.4 μm, respectively. In comparison, the protein coat of HIV is about 100 nm in diameter, and that of Qβ is about 26 nm, so the RNAs are 30 to 50 times longer than their viral protein coats. Both types of virus have linear, single-stranded RNA genomes. Some of the viral coat proteins are effectively RNA-binding proteins, and by binding to the genome they enforce a highly compacted folding arrangement of the RNA within the viral particle. An example can be seen in the tobacco mosaic virus (TMV), a pathogen of tobacco plants. The single-stranded RNA genome of TMV, 6,400 nucleotides long, is wound into a tight left-handed helix by its packaging within the rodlike helical protein coat (Figure 9-2a).

The genomes of DNA viruses vary greatly in size and form (see Table 8-1), but all are longer than the viral capsid heads that enclose them. Many viral DNAs are circular for at least part of their life cycle. During viral replication inside a host cell, specific types of viral DNA, called replicative forms, may appear; for example, many linear DNAs become circular, and all single-stranded DNAs become double-stranded. Bacteriophage T2 has a double-stranded linear DNA genome of 160,000 bp, a molecule more than 50 μm long that must be packaged into a virus head about 100 nm across in its longest dimension (Figure 9-2b). Bacteriophage φX174 is much smaller, a 5,386 nucleotide single-stranded circle (about 1.9 μm long) enclosed within a capsid 25 nm in diameter. Table 9-2 summarizes the genome and particle dimensions for several DNA viruses.

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Bacteria A single E. coli cell contains almost 100 times more DNA than a bacteriophage λ particle (see Table 9-2). The chromosome of the most common E. coli strain studied in laboratories worldwide (K-12 MG1655) is a single, double-stranded, circular DNA molecule (Table 9-3). Its 4,639,221 bp have a contour length of about 1.7 mm, some 850 times the length of the E. coli cell, 2 μm. In addition to the very large, circular DNA chromosome, many bacteria contain one or more plasmids, much smaller circular DNA molecules that are free in the cytosol (Figure 9-3; see also Chapter 7). Most plasmids are only a few thousand base pairs long, but some have more than 100,000 bp. Most do not encode genes essential to their host, but they are often symbiotic. They carry genetic information and undergo replication to yield daughter plasmids, which pass into the daughter cells at cell division. The spread of bacterial plasmids (and transposons) that confer antibiotic resistance among pathogenic bacteria has reduced the utility of standard antibiotics in medicine and agriculture (Highlight 9-1).

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Eukaryotes A yeast cell, one of the simplest eukaryotes, has 2.6 times more DNA in its genome than an E. coli cell (see Table 9-3). Cells of Drosophila melanogaster, the fruit fly used in classical genetics studies, contain more than 35 times as much DNA as E. coli cells, and human cells have almost 700 times as much. The cells of many plants and amphibians contain even more. All of this DNA must fit into a eukaryotic cell that is typically 10 to 20 μm across (although size can vary greatly, even within a single organism). The genetic material of eukaryotic cells is apportioned into multiple chromosomes, the diploid (2n) number depending on the species. A human somatic cell, for example, has 46 chromosomes (Figure 9-4). Each chromosome of a eukaryotic cell contains a single, very large, duplex DNA molecule. The DNA molecules in the 24 different types of human chromosomes (22 matching pairs plus the X and Y sex chromosomes) vary in length over a 25-fold range. Each type of chromosome in eukaryotes carries a characteristic set of genes.

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Eukaryotic cells also have organelles that contain DNA. Mitochondria and chloroplasts carry their own genomic DNAs (Figure 9-5). The evolutionary origin of mitochondrial and chloroplast DNAs has been the subject of much speculation. A widely accepted hypothesis, proposed by Lynn Margulis, is that they are vestiges of the chromosomes of ancient bacteria that gained access to the cytoplasm of host cells and became the precursors of these organelles (see Figure 8-17a). Mitochondrial DNA (mtDNA) codes for mitochondrial tRNAs and rRNAs and a few mitochondrial proteins; more than 95% of mitochondrial proteins are encoded by nuclear DNA. Mitochondria and chloroplasts divide when the cell divides. Their DNA is replicated before and during cell division, and the daughter DNA molecules pass into the daughter organelles.

Mitochondrial DNA molecules are much smaller than nuclear chromosomes. In animal cells, mtDNA contains fewer than 20 kbp (16,569 bp in human mtDNA) and is a circular duplex. Each mitochondrion typically has 2 to 10 copies of the mtDNA, but the number can be much higher: hundreds in muscle cells, and 100,000 in a mature oocyte. In a few organisms (e.g., trypanosomes, the parasites that cause sleeping sickness), the mitochondrial DNA is particularly abundant and organized. These mitochondria contain thousands of copies of mtDNA organized into a complex interlinked matrix known as a kinetoplast. Plant cell mtDNA is much larger than that in animal cells, ranging from 200 to 2,500 kbp. Chloroplast DNA (cpDNA) exists as circular duplexes of 120 to 160 kbp. Organelle DNA, like nuclear DNA, undergoes considerable compaction: DNA molecules 5 to 500 μm long must be accommodated in organelles about 1 to 5 μm in diameter.

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Some eukaryotes also contain plasmids; they have been found in yeast and some other fungi.