Chapter 10: Molecular Structure of Chromosomes and Transposable Elements PDF

CHAPTER OUTLINE 10.1 Organization of Functional Sites Along Prokaryotic Chromosomes 10.2 Structure of Prokaryotic Chromosomes 10.3 Organization of Functional Si...

CHAPTER OUTLINE 10.1 Organization of Functional Sites Along Prokaryotic Chromosomes 10.2 Structure of Prokaryotic Chromosomes 10.3 Organization of Functional Sites Along Eukaryotic Chromosomes 10.4 Sizes of Eukaryotic Genomes and Repetitive Sequences 10.5 Transposition 10.6 Structure of Eukaryotic Chromosomes in Nondividing Cells 0.8 μm 10.7 Structure of Eukaryotic 10 Chromosomes During Cell Division Structure of a bacterial chromosome. Electron micrograph of a bacte- rial chromosome, which has been released from a bacterial cell. Dr. Gopal Murti/Science Source MOLECULAR STRUCTURE OF CHROMOSOMES AND TRANSPOSABLE ELEMENTS Chromosomes are the structures within living cells that contain the organization of functional sites along a chromosome. Second, we genetic material. The term genome refers to the entire complement will examine the molecular structures of chromosomes. Third, we of genetic material in an organism or species. For prokaryotes, the will explore a process called transposition, in which short seg- genome is typically a single circular chromosome. For eukaryotes, ments of DNA, called transposable elements (TEs), are able to genetic material is found in different cellular compartments. The move to multiple sites within chromosomes and accumulate in nuclear genome in humans includes 22 autosomes, the X chromo- large numbers. Finally, we will examine the molecular mecha- some, and (in males) the Y chromosome. Eukaryotes also have a nisms that make chromosomes more compact. mitochondrial genome, and plants have a chloroplast genome. The primary function of the genetic material is to store the information needed to produce the characteristics of an organism. 10.1 ORGANIZATION OF As we saw in Chapter 9, the sequence of bases in a DNA molecule FUNCTIONAL SITES stores information. To fulfill their role at the molecular level, chro- ALONG PROKARYOTIC mosomal sequences facilitate four important processes: CHROMOSOMES ∙∙ the synthesis of RNA and cellular proteins ∙∙ Learning Outcome: the replication of chromosomes ∙∙ the proper segregation of chromosomes 1. Describe the general organization of functional sites along bacterial and archaeal chromosomes. ∙∙ the compaction of chromosomes so that they fit within liv- ing cells Both bacteria and archaea are considered prokaryotes. They are In this chapter, we will examine key features of prokaryotic mostly unicellular species that lack the complex cellular and eukaryotic chromosomes. First, we will consider the general compartmentalization found in eukaryotes. A hallmark characteristic 237 bro50795_ch10_237-269.indd 237 17/06/23 11:01 AM  238 C H A P T E R 1 0 :: MOLECULAR STRUCTURE OF CHROMOSOMES AND TRANSPOSABLE ELEMENTS FI GURE 10.1 Organization Origin of of sequences in prokaryotic replication chromosomal DNA. CONCEPT CHECK: What types of Key features: sequences constitute most of a prokaryotic genome? Most, but not all, prokaryotic species contain circular chromosomal DNA. Most prokaryotic species contain a single type of chromosome, but it may be present in multiple copies. A typical chromosome is a few million base pairs in length. Several thousand different genes are interspersed throughout the chromosome. The short regions between adjacent genes are called intergenic regions. At least one origin of replication is required to initiate DNA replication. Genes Intergenic regions Repetitive sequences may be interspersed throughout the chromosome. Repetitive sequences of prokaryotic cells is that they lack a cell nucleus that is bounded by sequences sometimes play a role in a variety of genetic a double membrane. processes, including DNA folding, gene regulation, and Figure 10.1 shows the general features of a prokaryotic genetic recombination. As discussed in Section 10.5, some chromosome. repetitive sequences are transposable elements that can move throughout the genome. ∙∙ In most species, the chromosomal DNA is a circular mole- cule, though some species have linear chromosomes (Fig- ure 10.1). Although bacteria and archaea usually contain a single type of chromosome, more than one copy of that 10.1 COMPREHENSION QUESTION chromosome may be found within one cell. 1. A bacterial chromosome typically contains ∙∙ A typical prokaryotic chromosome is a few million base pairs a. a few thousand genes. (bp) in length. For example, the chromosome of Escherichia b. one origin of replication. coli has approximately 4.6 million bp, and that of Haemophi- lus influenzae has roughly 1.8 million bp. A prokaryotic chro- c. some repetitive sequences. mosome commonly has a few thousand different genes, d. all of the above. which are interspersed throughout the entire chromosome. Protein-coding genes account for the majority of the chro- mosomal DNA. The nontranscribed regions of DNA located 10.2 STRUCTURE OF between adjacent genes are termed intergenic regions. PROKARYOTIC ∙∙ Bacterial chromosomes usually have one origin of repli- cation as do some archaeal species (see Figure 10.1), CHROMOSOMES though other archaea have multiple origins. The origin is a Learning Outcomes: DNA sequence that is a few hundred base pairs in length. 1. Outline the processes that make a prokaryotic chromosome This nucleotide sequence functions as an initiation site for more compact. the assembly of several proteins required for DNA replica- 2. Describe how DNA gyrase causes DNA supercoiling. tion. However, the mechanism for how DNA replication is initiated in bacteria is different from how it starts in ar- chaea (see Chapter 11). Inside a prokaryotic cell, a chromosome is highly compacted and ∙∙ A wide variety of repetitive sequences have been identi- found within a region of the cell known as a nucleoid. Depending fied in prokaryotic species. These sequences are found in on the growth conditions and phase of the cell cycle, prokaryotes multiple copies and are usually interspersed within the in- typically have one to four identical chromosomes per cell. In addi- tergenic regions throughout the chromosome. Repetitive tion, the number of copies varies depending on the species. Having bro50795_ch10_237-269.indd 238 17/06/23 11:01 AM  10.2 Structure of Prokaryotic Chromosomes 239 0.3 μm FI GURE 10.2 The localization of nucleoids within the bacte- rium Bacillus subtilis. The nucleoids are fluorescently labeled, so they appear as bright blue regions within the bacterial cytoplasm. Note that two or more nucleoids may be found within a cell. M. Wurtz/Biozentrum, University of Basel/Science Source CONCEPT CHECK: How many nucleoids are in this bacterial cell? multiple nucleoids may enhance a cell’s ability to synthesize more proteins. Figure 10.2 is a micrograph in which the nucleoids are fluo- rescently labeled in blue. Each chromosome is found within its F I G URE 1 0. 3 Core and microdomains of a bacterial chro- mosome. This is a schematic drawing of an E. coli chromosome that own distinct nucleoid in the cell. Unlike the eukaryotic nucleus, has been extracted from a cell and viewed by electron microscopy. The the nucleoid is not a separate cellular compartment surrounded by core is in the center with many loops (microdomains) emanating from it. a membrane. Rather, the DNA in a nucleoid is in direct contact Not all bacterial species have their chromosomes organized into micro- with the cytoplasm of the cell. In this section, we will explore the domains and macrodomains. structure of prokaryotic chromosomes and the processes by which Source: Wang, Xindan, Llopis, Paula Montero, and Rudner, David Z., “Organization and they are compacted to fit within a nucleoid. Segregation of Bacterial Chromosomes,” Nature Reviews Genetics, vol. 14, no. 3, March, 2013, 191–203. The Formation of Chromosomal Loops Helps regions. The existence of macrodomains was first deter- Make the Bacterial Chromosome More Compact mined by measuring the frequency of recombination To fit within the average-sized prokaryotic cell, the chromosomal (crossing over) between particular sites scattered through- DNA must be compacted about 1000-fold. The mechanism of bac- out the E. coli chromosome. Recombination is much more terial chromosome compaction is not entirely understood, and it frequent between sites within a macrodomain than be- may vary among different species. Figure 10.3 shows a schematic tween sites in different macrodomains. Therefore, macro- drawing of a chromosome that has been removed from an E. coli domains are also called chromosomally interacting cell. As the drawing shows, the chromosome has a central core domains (CIDs). Because the identification of macrodo- with many loops emanating from the core. mains is not based on electron microscopy, they are not evident in Figure 10.3. ∙∙ Based on microscopy studies, the loops that emanate from the core, which are called microdomains, are typically To form microdomains and macrodomains, bacteria use a 10,000 base pairs (10 kbp) in length. An E. coli chromo- set of DNA-binding proteins called nucleoid-associated proteins some is expected to have about 400 to 500 microdomains. (NAPs) that facilitate chromosome compaction and organization. The lengths and boundaries of these microdomains are These proteins either bend the DNA or act as bridges that cause thought to be dynamic, changing in response to environ- different regions of DNA to bind to each other. NAPs also facili- mental conditions. tate chromosome segregation and play a role in gene regulation. ∙∙ In E. coli, many adjacent microdomains are further orga- Examples of NAPs include histone-like nucleoid structuring nized into macrodomains that are about 800 to 1000 kbp (H-NS) proteins and structural maintenance of chromosomes in length; each macrodomain contains about 80 to 100 mi- (SMC) proteins. SMCs are also found in eukaryotes, and later in crodomains. One proposed model suggests that the E. coli this chapter, we will examine how they tether segments of DNA to chromosome has 4 macrodomains and two nonstructured each other (look ahead to Figure 10.21). bro50795_ch10_237-269.indd 239 17/06/23 11:01 AM  240 C H A P T E R 1 0 :: MOLECULAR STRUCTURE OF CHROMOSOMES AND TRANSPOSABLE ELEMENTS GENETIC TIPS THE QUESTION: As noted in Chapter 9, 1 bp of DNA is approximately 0.34 nm in length. A bacterial chromosome is about 4 million bp in length. The dimensions of the cytoplasm of a bacterium, such as E. coli, are roughly 0.5 µm wide Loop and 1.0 µm long. domain A. A  microdomain is a loop of DNA that contains about 10,000 bp. If it was stretched out linearly, how long (in micrometers) would a microdomain be? B. If a bacterial microdomain was circular, what would be its diameter? (Note: Circumference = πD, where D is the diameter of the circle.) Nucleosome composed of Alba protein 60 bp of DNA wrapped around C. Is the diameter of the circular microdomain calculated in part B a histone tetramer. small enough to fit inside a bacterium? F I G URE 1 0. 4 Chromosome structure in some archaea. This T OPIC: What topic in genetics does this question address? The illustration shows a short region of an archaeal chromosome. In this exam- topic is the dimensions of a bacterial chromosome. More ple, 60 bp of DNA is wrapped around a histone tetramer to form a nucleo- specifically, the question asks you to calculate the dimensions of a some. The repeating occurrence of nucleosomes resembles beads on a string. Loop domains are created by the Alba protein, which forms a bridge microdomain. between two different segments of DNA. Though not shown in this figure, I NFORMATION: What information do you know based on the the DNA is also supercoiled to make the chromosome more compact. question and your understanding of the topic? In the question, you are reminded that the length of 1 bp of DNA is about 0.34 nm By contrast, other archaeal species produce eukaryotic-like and that a bacterial chromosome is about 4 million bp in length. histone proteins. (Histones are described in Section 10.6.) In One microdomain is a loop with about 10,000 bp. You are also these species, the DNA is wrapped around histone proteins to told that the bacterial cytoplasm is about 0.5 µm wide and 1.0 µm form nucleosomes and also organized into loop domains by a long and given the equation for calculating the circumference of protein called Alba (Figure 10.4). The number of histone pro- a circle. teins within archaeal nucleosomes varies among different spe- P ROBLEM-SOLVING S TRATEGIES: Make a calculation. cies. In some archaea, 60 bp of DNA is wrapped around a histone Compare and contrast. For part A, you simply multiply 10,000 tetramer to form a “beads-on-a-string” structure (see Fig- by 0.34 nm, which is the length of 1 bp. For part B, you use the ure 10.19a). This structure is similar to eukaryotic chromatin ex- equation that is given. The circumference is the linear length of cept that eukaryotic nucleosomes contain histone octamers (look the DNA. For part C, you compare the answer to part B to the ahead to Figure 10.17). dimensions of the bacterial cytoplasm. DNA Supercoiling Further Compacts ANSWER: a Prokaryotic Chromosome A. One microdomain is 10,000 bp. One base pair is 0.34 nm, which equals 0.00034 µm. You multiply these two numbers: Because DNA is a long thin molecule, twisting forces can dra- (10,000) (0.00034 µm) = 3.4 µm matically change its conformation. This effect is similar to what happens when you twist a rubber band. If twisted in one direction, B. Circumference = πD a rubber band eventually coils itself into a compact structure as it 3.4 µm = πD absorbs the energy applied by the twisting motion. Because the D = 1.1 µm two strands within DNA already coil around each other, the for- C. The diameter is a little too big to fit inside a bacterium such as mation of additional coils due to twisting forces is referred to as E. coli. NAPs and supercoiling make the microdomains much DNA supercoiling. The DNA within microdomains and loop do- more compact so that a single chromosome can occupy a nucleoid mains is further compacted because of DNA supercoiling. within the bacterial cell. How do twisting forces affect DNA structure? Figure 10.5 illustrates four possibilities. In Figure 10.5a, a double-stranded DNA molecule with five complete turns is anchored between two plates. In this hypothetical example, the ends of the DNA Some Archaeal Chromosomes Contain Histone molecule cannot rotate freely. Both underwinding and over- Proteins that Form Nucleosomes winding of the DNA double helix can cause supercoiling of the The structure of the archaeal chromosome varies among different helix. As described in Chapter 9, the predominant form of DNA, species depending on the DNA-binding proteins they express. called B DNA, is a right-handed helix. Therefore, underwinding Some archaeal species produce bacterial-like nucleoid-associated is a left-handed twisting motion, and overwinding is a right- proteins. These species organize their chromosomes in a way that handed twisting motion. Along the left side of Figure 10.5, one appears to be similar to bacterial species. (see Figure 10.3). of the plates has been given a left-handed twist that tends to bro50795_ch10_237-269.indd 240 17/06/23 11:01 AM  10.2 Structure of Prokaryotic Chromosomes 241 FIGURE 10.5 Schematic representation of DNA supercoiling. In this example, the DNA in 1 (a) is anchored between two plates and given a twist 2 as noted by the arrows. A left-handed twist (under- 10 bp 3 winding) can produce either (b) fewer turns or (c) a per turn negative supercoil. A right-handed twist (overwinding) can pro- 4 duce (d) more turns or (e) a positive supercoil. The structures 5 shown in (b) and (d) are unstable. (a) No supercoil 360° left-handed 360° right-handed twist (underwinding) twist (overwinding) or or 1 1 2 12.5 bp 10 bp 8.3 bp 2 10 bp 1 1 per turn 2 per turn per turn 3 per turn 3 (not a plus 1 (not a plus 1 2 4 4 stable 3 negative 3 stable positive structure) supercoil 5 structure) 5 supercoil 5 4 4 6 (b) Unstable (c) Negative supercoil (d) Unstable (e) Positive supercoil unwind the helix. As the helix absorbs this force, the under- supercoiling is due to an underwinding force on the DNA. There- winding can cause: fore, negative supercoiling creates tension on the DNA strands that may be released by their separation (Figure 10.6). Although ∙∙ fewer turns (Figure 10.5b) or most of the chromosomal DNA is negatively supercoiled and ∙∙ the formation of a negative supercoil (Figure 10.5c). compact, the force of negative supercoiling may promote DNA On the right side of Figure 10.5, one of the plates has been given strand separation in small regions. This enhances genetic activities a right-handed twist, which overwinds the double helix. Over- such as replication and transcription that require the DNA strands winding can cause: to be separated. How does DNA become supercoiled? In 1976, Martin ∙∙ more turns (Figure 10.5d) or Gellert and colleagues discovered the enzyme DNA gyrase, also ∙∙ the formation of a positive supercoil (Figure 10.5e). known as topoisomerase II. This enzyme, which contains four The DNA conformations shown in Figure 10.5a, c, and e differ subunits (two A and two B subunits), introduces negative super- only with regard to supercoiling. These three DNA conformations coils (or relaxes positive supercoils) using energy from are referred to as topoisomers of each other. The DNA conforma- tions shown in Figure 10.5b and d are not structurally stable and Area of do not occur in living cells. negative supercoiling Chromosome Function Is Influenced by Strand DNA Supercoiling separation The chromosomal DNA in living bacteria and archaea is nega- tively supercoiled. For example, in the chromosome of E. coli, about one negative supercoil occurs per 40 turns of the double helix. Negative supercoiling has important consequences. As al- ready mentioned, the supercoiling of chromosomal DNA makes it Circular much more compact. Therefore, supercoiling helps to greatly de- chromosome crease the size of a prokaryotic chromosome. In addition, negative supercoiling also affects DNA func- FIGURE 10.6 Negative supercoiling promotes strand separation. tion. To understand how it does so, remember that negative CONCEPT CHECK: Why is strand separation beneficial? bro50795_ch10_237-269.indd 241 17/06/23 11:01 AM  242 C H A P T E R 1 0 :: MOLECULAR STRUCTURE OF CHROMOSOMES AND TRANSPOSABLE ELEMENTS Upper jaws DNA wraps around DNA binds to the A subunits in a Upper jaws the lower jaws. right-handed direction. clamp onto DNA. DNA held in lower jaws is cut. DNA held in upper Lower jaws DNA jaws is released and passes downward through the opening A subunits B subunits in the cut DNA. This process uses 2 ATP molecules. (a) Molecular mechanism of DNA gyrase function Circular Cut DNA is ligated back DNA 2 negative together and the DNA is molecule DNA gyrase supercoils released from DNA gyrase. 2 ATP (b) Overview of DNA gyrase function FIGURE 10.7 The action of DNA gyrase. (a) DNA gyrase, also known as topoisomerase II, is composed of two A and two B subunits. The lower jaws (shown in red) first bind to the DNA, and then the DNA forms a loop and a second site in the DNA binds to the upper jaws (shown in green). The DNA in the lower jaws is then cut (i.e., a double-stranded break is made). The unbroken segment of DNA is released from the upper jaws and passes through the break. The break is repaired. The B subunits capture the energy from the hydrolysis of two ATP molecules to catalyze this process. (b) The result is that two negative turns have been introduced into the DNA molecule. CONCEPT CHECK: In your own words, describe the step that requires the use of ATP. ATP (Figure 10.7a). To alter supercoiling, DNA gyrase has two region and introduce a break in one of the DNA strands. After sets of jaws that allow it to grab onto two regions of DNA. one DNA strand has been broken, the DNA molecule rotates to relieve the tension that is caused by negative supercoiling. 1. To begin the process, a DNA region is grabbed by the This rotation relaxes negative supercoiling. The broken strand lower jaws. is then repaired. The competing actions of DNA gyrase and 2. The DNA is then wrapped in a right-handed direction topoisomerase I govern the overall supercoiling of the bacte- around the two A subunits. rial DNA. 3. The upper jaws then clamp onto another region of DNA. The ability of DNA gyrase to introduce negative supercoils 4. The DNA in the lower jaws is cut in both strands, and the into DNA is critical for bacterial (and archaeal) cell survival. other region of DNA is then released from the upper jaws Therefore, much research has been aimed at identifying drugs that and passed through this double-stranded break. specifically block this enzyme’s function as a way to cure or alle- 5. To complete the process, the double-stranded break is ligated viate diseases caused by bacteria. Two main classes—quinolones back together. The net result is that two negative supercoils and coumarins—inhibit gyrase and other bacterial topoisomer- have been introduced into the DNA molecule (Figure 10.7b). ases, thereby blocking bacterial cell growth. These drugs do not In addition, DNA gyrase can untangle DNA molecules. For exam- inhibit eukaryotic topoisomerases, which are structurally different ple, as discussed in Chapter 11, circular DNA molecules are some- from their bacterial counterparts. This finding has been the basis times intertwined following DNA replication (see Figure 11.14). for the production of many drugs with important antibacterial ap- Such interlocked molecules can be separated by DNA gyrase. plications. An example is ciprofloxacin (known also by the brand A second type of enzyme, topoisomerase I, relaxes nega- name Cipro), which is used to treat a wide spectrum of bacterial tive supercoils. This enzyme can bind to a negatively supercoiled diseases, including anthrax. bro50795_ch10_237-269.indd 242 17/06/23 11:01 AM  10.3 Organization of Functional Sites Along Eukaryotic Chromosomes 243 ∙∙ Origins of replication are chromosomal sites that are neces- 10.2 COMPREHENSION QUESTIONS sary to initiate DNA replication. Unlike most bacterial 1. Mechanisms that make the bacterial chromosome more com- ­chromosomes, which contain only one origin of replication, pact include eukaryotic chromosomes contain many origins, interspersed a. the formation of microdomains and macrodomains. approximately every 100,000 bp. The function of origins of replication is discussed in greater detail in Chapter 11. b. DNA supercoiling. ∙∙ Centromeres are regions that play a role in the proper c. crossing over. segregation of chromosomes during mitosis and meiosis. d. both a and b. In most eukaryotic species, each chromosome contains a 2. Negative supercoiling can enhance RNA transcription and DNA single centromere, which usually appears as a constricted replication because it region of a mitotic chromosome. A centromere functions a. allows the binding of proteins in the major groove. as a site for the formation of a kinetochore, which b. promotes DNA strand separation. c. makes the DNA more compact. Telomere Key features: d. causes all of the above. Eukaryotic chromosomes are usually linear. 3. DNA gyrase a. promotes negative supercoiling. Eukaryotic chromosomes occur in sets. Many species are diploid, which means that b. relaxes positive supercoils. somatic cells contain 2 sets of chromosomes. Origin of c. cuts DNA strands as part of its function. replication A typical chromosome is tens of millions to d. does all of the above. hundreds of millions of base pairs in length. Genes are interspersed throughout the chromosome. A typical chromosome 10.3 ORGANIZATION OF contains between a few hundred and several FUNCTIONAL SITES Origin of replication thousand different genes. ALONG EUKARYOTIC Each chromosome contains many origins of replication that are interspersed about every CHROMOSOMES Kinetochore proteins 100,000 base pairs. Learning Outcome: Centromere Each chromosome contains a centromere that forms a recognition site for the 1. Describe the organization of functional sites along a eukary- kinetochore proteins. otic chromosome. Telomeres contain specialized sequences located at both ends of the linear chromosome. Figure 10.8 shows the general features of a eukaryotic chromo- Origin of replication some and the functional sites along it. Repetitive sequences are commonly found near centromeric and telomeric regions, but ∙∙ Each eukaryotic chromosome contains a long, linear DNA they may also be interspersed throughout molecule. the chromosome. ∙∙ Eukaryotic species have one or more sets of chromosomes in the cell nucleus; each set is composed of several differ- ent linear chromosomes (refer back to Figure 8.1). Hu- Origin of replication mans, for example, have two sets of 23 chromosomes each, for a total of 46. ∙∙ A typical eukaryotic chromosome is typically tens of mil- lions to hundreds of millions of base pairs in length. ∙∙ A single chromosome usually carries hundreds to several thousand different genes. A typical eukaryotic gene is sev- Telomere Genes eral thousand to tens of thousands of base pairs in length. In Repetitive sequences less complex eukaryotes such as yeast, genes are relatively small, often several hundred to a few thousand base pairs FIGURE 10.8 General features of a eukaryotic chromosome. long. In more complex eukaryotes such as mammals and Note: This is meant to be a schematic representation that depicts a flowering plants, protein-coding genes tend to be much lon- metaphase chromosome. The genetic elements are not drawn to scale. ger due to the presence of introns—noncoding intervening The numbers of origins of replication, genes, and repetitive sequences sequences. The size of introns ranges from less than 100 bp are much higher than shown here. to more than 10,000 bp. Therefore, the presence of large CONCEPT CHECK: What are some differences between the types of sequences introns can greatly increase the lengths of eukaryotic genes. found in eukaryotic chromosomes and those in prokaryotic chromosomes? bro50795_ch10_237-269.indd 243 17/06/23 11:01 AM  244 C H A P T E R 1 0 :: MOLECULAR STRUCTURE OF CHROMOSOMES AND TRANSPOSABLE ELEMENTS assembles just before and during the very early stages of mitosis and meiosis. 10.4 SIZES OF EUKARYOTIC In certain yeast species, such as Saccharomyces cere- GENOMES AND visiae, the centromere has a defined DNA sequence that is REPETITIVE SEQUENCES about 125 bp in length. This type of centromere is called a point centromere. By comparison, the centromeres found in Learning Outcomes: more complex eukaryotes are much larger and contain tan- 1. Describe the variation in size of eukaryotic genomes. dem arrays of short repetitive DNA sequences. (Tandem 2. Define repetitive sequence, and explain how this type of arrays are described in Section 10.4.) These are called re- sequence affects genome sizes. gional centromeres. They can range in length from several thousand to more than a million base pairs. By themselves, the repeated DNA sequences within regional centromeres The total amount of DNA in cells of eukaryotic species is usually are not necessary or sufficient to form a functional centro- much greater than the amount in prokaryotic cells. In addition, mere with a kinetochore. Other features must be present in a eukaryotic genomes contain many more genes than their prokary- functional centromere. For example, a distinctive feature of otic counterparts. In this section, we will examine the sizes of eu- all eukaryotic centromeres is that histone protein H3 is re- karyotic genomes and consider how repetitive sequences may placed with a histone variant called CENP-A. (Histone vari- have a significant effect on those sizes. ants are described in Chapter 15.) However, researchers are still trying to identify all of the biochemical properties that The Sizes of Eukaryotic Genomes Vary distinguish regional centromeres and understand how these Substantially properties are transmitted during cell division. Different eukaryotic species vary dramatically in the size of their ∙∙ The kinetochore is composed of a group of proteins that genomes (Figure 10.9a; note that the graph uses a log scale). In link the centromere to the spindle apparatus during mitosis many cases, this variation is not related to the complexity of the and meiosis, ensuring the proper segregation of the chro- species. For example, two closely related species of salamander, mosomes to each daughter cell. Plethodon richmondi and Plethodon larselli, differ considerably in ∙∙ The ends of linear eukaryotic chromosomes have special- genome size (Figure 10.9b, c). The genome of P. larselli is more ized regions known as telomeres. Telomeres serve several than twice as large as the genome of P. richmondi. However, the important functions in the replication and stability of the genome of P. larselli probably doesn’t contain more genes. How chromosome. As discussed in Chapter 8, telomeres pre- do we explain the difference in genome size? The additional DNA vent chromosomal rearrangements such as translocations. in P. larselli is due to the accumulation of many copies of repeti- In addition, they prevent chromosome shortening in two tive DNA sequences. In some species, the amounts of these re- ways. First, the telomeres protect chromosomes from di- petitive sequences have reached enormous levels. Such repetitive gestion via enzymes called exonucleases that recognize the sequences do not code proteins, and their function remains a mat- ends of DNA. Second, a specialized form of DNA replica- ter of controversy and great interest. The structure and signifi- tion occurs at the telomeres so that eukaryotic chromo- cance of repetitive DNA sequences are discussed next. somes do not become shortened with each round of DNA replication (see Chapter 11). However, shortening does The Genomes of Eukaryotes Contain Sequences occur in adult somatic cells as a part of the aging process. That Are Unique, Moderately Repetitive, or Highly Repetitive 10.3 COMPREHENSION QUESTIONS The term sequence complexity refers to the number of times a particular base sequence appears throughout the genome of a spe- 1. The chromosomes of eukaryotes typically contain cies. Unique, or nonrepetitive, sequences are those found once or a. a few hundred to several thousand different genes. a few times within a genome. Protein-coding genes are typically b. multiple origins of replication. unique sequences of DNA. The vast majority of proteins in eu- c. a centromere. karyotic cells are coded by genes present in one or a few copies. In d. telomeres at their ends. the case of humans, unique sequences make up roughly 41% of the e. all of the above. entire genome (Figure 10.10). These unique sequences include the protein-coding regions of genes (2%), introns (24%), and 2. The kinetochore is attached to ______ and its function is to unique regions that are not found within genes (15%). ______. Moderately repetitive sequences are found a few hundred a. a gene, promote transcription to several thousand times in a genome. In a few cases, moderately b. the centromere, promote chromosome segregation during repetitive sequences are multiple copies of the same gene. For mitosis and meiosis example, the genes that code ribosomal RNA (rRNA) are found in c. a telomere, prevent chromosome shortening many copies. Ribosomal RNA is necessary for the functioning of d. the centromere, promote chromosome replication ribosomes. Cells need a large amount of rRNA for making bro50795_ch10_237-269.indd 244 17/06/23 11:01 AM  10.4 Sizes of Eukaryotic Genomes and Repetitive Sequences 245 Fungi Vascular plants Insects Mollusks Fishes (b) Plethodon richmondi Salamanders Amphibians Reptiles Birds Mammals 106 107 108 109 1010 1011 1012 (a) Genome sizes (nucleotide base pairs per haploid genome) (c) Plethodon Iarselli FI GURE 10.9 Haploid genome sizes among groups of eukaryotic species. (a) Ranges of genome sizes among different groups of eukary- otes. (b) A species of salamander, Plethodon richmondi, and (c) a close relative, Plethodon larselli. The genome of P. larselli is more than twice as large as that of P. richmondi. Genes→Traits The two species of salamander shown here have very similar traits, even though the genome of P. larselli is more than twice as large as that of P. richmondi. However, the genome of P. larselli is not likely to contain twice as many genes. Rather, the additional DNA is due to the accumulation of short repetitive DNA sequences that do not contain functional genes and are present in many copies. (a): Source: Gregory, T. Ryan, “Eukaryotic Genome Size Databases,” Nucleic Acids Research, vol. 35, January, 2007, D332–D338.; (b): Ann & Rob Simpson; (c): Gary Nafis CONCEPT CHECK: What are two reasons for the wide variation in genome sizes among eukaryotic species? ribosomes, and producing such an amount is facilitated by having sequences may play a role in the regulation of gene transcription multiple copies of the genes that code rRNA. Likewise, the genes and translation. By comparison, some moderately repetitive se- that code histone proteins are also found in multiple copies be- quences do not play a functional role and are derived from trans- cause a large number of histone proteins are needed for the struc- posable elements (TEs)—short segments of DNA that have the ture of chromosomes. ability to move within a genome. This category of repetitive se- In addition, other types of functionally important sequences quences is discussed in greater detail in Section 10.5. are moderately repetitive. For example, moderately repetitive Highly repetitive sequences are found tens of thousands or even millions of times throughout a genome. Each copy of a highly 100 repetitive sequence is relatively short, ranging from a few nucleotides Percentage in the human genome to several hundred in length. A widely studied example is the Alu fam- 80 ily of sequences found in humans and other primates. The Alu se- quence is approximately 300 bp long. This sequence derives its name 59% from the observation that it contains a site for cleavage by a restriction 60 Unique sequences enzyme known as AluI. (The function of restriction enzymes is de- 40 scribed in Chapter 20.) The Alu sequence represents about 10% of the 24% total human DNA and occurs approximately every 5000–6000 bp! 20 15% Evolutionary studies suggest that the Alu sequence arose 65 mya from a section of a single ancestral gene known as 7SL RNA. Since that 2% 0 time, this gene has become a type of TE called a retrotransposon, Regions of Introns and Unique Repetitive which is transcribed into RNA, copied into DNA, and inserted into genes that other parts sequences DNA code of genes not found the genome (see Section 10.5). Over the past 65 million years, the Alu proteins such as within genes sequence has been copied and inserted into the human genome many (exons) enhancers times and is now present in about 1,000,000 copies. Classes of DNA sequences Repetitive sequences, like those of the Alu family, are inter- spersed throughout the genome. However, some moderately and FI GURE 10.10 Relative amounts of unique and repetitive highly repetitive sequences are clustered together in a tandem array, DNA sequences in the human genome. also known as a tandem repeat. In a tandem array, a very short bro50795_ch10_237-269.indd 245 17/06/23 11:01 AM  246 C H A P T E R 1 0 :: MOLECULAR STRUCTURE OF CHROMOSOMES AND TRANSPOSABLE ELEMENTS nucleotide sequence is repeated many times in a row. In Drosophila, enable them to be mobile. In this section, we will examine the for example, 19% of the chromosomal DNA consists of highly repeti- characteristics of TEs and explore the mechanisms by which they tive sequences found in tandem arrays. An example is shown here. move. We will also discuss the biological significance of TEs. AATATAATATAATATAATATAATATATAATAT TTATATTATATTATATTATATTATATATTATA McClintock Found That Chromosomes of Corn Plants Contain Loci That Can Move In this particular tandem array, two related sequences, AATAT and AATATAT (in the top strand), are repeated. As mentioned McClintock’s scientific work was focused on the structure and earlier, tandem arrays of short sequences are commonly found in function of the chromosomes of corn plants. This research involved centromeric regions of chromosomes and can be quite long, some- countless hours of examining corn chromosomes under the micro- times more than 1,000,000 bp in length! scope. McClintock was technically gifted and had a theoretical What is the functional significance of highly repetitive se- mind that could propose ideas that conflicted with conventional quences? Whether they have any significant function is controver- wisdom. sial. Some experiments in Drosophila indicate that highly McClintock identified many unusual features of chromo- repetitive sequences may be important in the proper segregation of somes in different strains of corn. In one strain, a particular site in chromosomes during meiosis. It is not yet clear if highly repetitive chromosome 9 had the strange characteristic of showing a fairly DNA plays the same role in other species. The sequences within high rate of breakage. McClintock termed this a mutable site, or highly repetitive DNA vary greatly from species to species. Like- mutable locus. The mutable locus was named Ds (for dissocia- wise, the amount of highly repetitive DNA can vary a great deal tion), because chromosomal breakage occurred frequently there. even among closely related species (as noted in Figure 10.9). McClintock identified strains of corn in which the Ds locus was found in different places within the corn genome. In one case, Ds was located in the middle of a gene affecting kernel color. The C allele provides dark red color, whereas c is a recessive allele 10.4 COMPREHENSION QUESTION of the same gene and causes a colorless kernel. The endosperm of 1. Which of the following is/are moderately repetitive sequences? corn kernels is triploid. The drawing below shows the genotype of a. Genes that code rRNA chromosome 9 in the endosperm of one of McClintock’s strains. b. Most protein-coding genes CDsC c. Both a and b d. None of the above c 10.5 TRANSPOSITION c Learning Outcomes: This strain had an interesting phenotype. Most of the corn 1. Summarize the studies of McClintock, and explain how they kernel was colorless, but it also contained some red sectors. revealed the existence of transposable elements. McClintock explained this phenotype in the following way: 2. Describe the organization of sequences within different types of transposable elements. 1. The colorless background of a kernel was due to the trans- 3. Explain how transposons and retrotransposons move to new position of Ds into the C allele, which would inactivate that locations in a genome. allele. 4. Discuss the effects of transposable elements on gene function. 2. In a few cells, Ds occasionally transposed out of the C allele during kernel growth (see drawing below). During transposition, Ds moved out of the C allele to a new As we have seen, sizeable portions of many species’ genomes are location, and the two parts of the C allele were rejoined, composed of repetitive sequences. In many cases, the repetitive thereby restoring its function. As the kernel grew, such a sequences are due to transposition, the process in which a DNA cell would continue to divide, resulting in a red sector. segment is inserted into a new location in the genome. The DNA segments that transpose themselves are known as transposable el- Ds has transposed out of C gene ements (TEs). TEs have sometimes been referred to as “jumping to a new chromosomal location. genes” because they are inherently mobile. Transposable elements were first identified by Barbara C McClintock in the early 1950s during classic studies with corn plants. Since that time, geneticists have discovered many different c types of TEs in organisms as diverse as bacteria, archaea, fungi, plants, and animals. The advent of molecular techniques has al- c lowed scientists to better understand the characteristics of TEs that bro50795_ch10_237-269.indd 246 17/06/23 11:01 AM  10.5 Transposition 247 On rare occasions, when McClintock crossed a strain carrying Ds in the middle of the C allele to a strain carrying the recessive c al- lele, the cross produced a kernel that was completely red. In this case, Ds had transposed out of the C allele prior to kernel growth, probably during gamete formation. In offspring that grew from a solid red kernel, McClintock determined that the Ds locus had Transposon moved out of the C allele to a new location. In addition, the re- stored C allele behaved normally. In other words, the C allele was no longer mutable; the kernels did not show a sectoring phenotype. Taken together, the results were consistent with the hypothesis that the Ds locus can move around the corn genome by transposition. Transposon When McClintock published these results in 1951, they were met with great skepticism. Some geneticists of that time were unable to accept the idea that the genetic material was susceptible (a) Simple transposition to frequent rearrangement. Instead, they believed that the genetic material was very stable and permanent in its structure. Over the next several decades, however, the scientific community came to realize that TEs are a widespread phenomenon. McClintock was Retrotransposon awarded the Nobel Prize in Physiology or Medicine in 1983, more Transcription than 30 years after the original discovery of transposable elements. DNA RNA Reverse transcriptase Transposable Elements Move by Different Transposition Pathways Since McClintock’s pioneering studies, many different TEs have been found in bacteria, archaea, fungi, plants, and animals. Two main types of transposition mechanisms have been identified: simple transposition and retrotransposition. Retrotransposon Retrotransposon Simple Transposition. In simple transposition, the TE is re- moved from its original site and transferred to a new target site (b) Retrotransposition (Figure 10.11a). This mechanism is called a cut-and-paste mecha- nism because the element is cut out of its original site and pasted F I G URE 1 0. 1 1 Different mechanisms of transposition. into a new one. Transposable elements that move via simple trans- CONCEPT CHECK: Which of these mechanisms causes the TE to increase position are widely found in bacterial and eukaryotic species. in number? Such TEs are also called transposons. Retrotransposition. Another type of transposable element also called target-site duplications, which are identical base moves via an RNA intermediate. This form of transposition, sequences that are oriented in the same direction and repeated. termed retrotransposition, is found only in eukaryotic species, Direct repeats are adjacent to both ends of any TE. where it is very common (Figure 10.11b). Transposable elements that move via retrotransposition are known as retrotransposons, Insertion Elements. The simplest TE is known as an insertion or retroelements. In retrotransposition, the element is transcribed element (IS element). As shown in Figure 10.12a, an IS element into RNA. An enzyme called reverse transcriptase uses the RNA has two important characteristics. First, both ends of the element as a template to synthesize a DNA molecule that is integrated into contain inverted repeats (IRs). Inverted repeats are DNA se- a new region of the genome. Retrotransposons increase in number quences that are identical (or very similar) but run in opposite di- during retrotransposition. rections, such as the following: Each Type of Transposable Element Has a 5’–CTGACTCTT–3’ and 5’–AAGAGTCAG–3’ Characteristic Pattern of DNA Sequences 3’–GACTGAGAA–5’ 3’–TTCTCAGTC–5’ Research on TEs from many species has established that the DNA Depending on the particular IS element, the inverted repeats sequences within them are organized in several different ways. range from 9 to 40 bp in length. In addition, IS elements may con- Figure 10.12 presents a few of those ways, although many varia- tain a central region that codes the enzyme transposase, which tions are possible. All TEs are flanked by direct repeats (DRs), catalyzes the transposition event. bro50795_ch10_237-269.indd 247 17/06/23 11:01 AM  248 C H A P T E R 1 0 :: MOLECULAR STRUCTURE OF CHROMOSOMES AND TRANSPOSABLE ELEMENTS By comparison, non-LTR retrotransposons do not resemble retroviruses in having LTRs. They may contain a gene that codes a protein that functions as both a reverse transcriptase and an endonucle- DR IR Transposase IR DR gene ase (see Figure 10.12b). As discussed later, these functions are needed Insertion element for retrotransposition. Some non-LTR retrotransposons are evolution- arily derived from normal eukaryotic genes. For example, the Alu family of repetitive sequences found in humans is derived from a single ancestral gene known as 7SL RNA (that codes a component of DR IR Transposase Antibiotic- IR DR the complex called signal recognition particle, which targets newly gene resistance made proteins to the endoplasmic reticulum). This gene sequence has Simple transposon gene been copied via retrotransposition many times, and the current number of copies in the human genome is approximately 1 million. (a) Elements that move by simple transposition Transposable elements are considered to be complete elements, or autonomous elements, when they contain all of the information necessary for transposition or retrotransposition to take place. How- DR LTR Reverse Integrase LTR DR ever, TEs are often incomplete, or nonautonomous. A nonautono- transcriptase gene mous element typically lacks a gene such as one that codes transposase gene or reverse transcriptase, which is necessary for transposition to occur. LTR retrotransposon The Ds locus, which is the mutable site in corn discussed previously, is a nonautonomous element, because it lacks a trans- posase gene. An element that is similar to Ds but contains a func- tional transposase gene is called the Ac element, which stands for DR Reverse DR transcriptase/ activator element. An Ac element provides a transposase gene that endonuclease enables Ds to transpose. Therefore, nonautonomous TEs such as Ds gene can transpose only when an Ac element is present at another region Non-LTR retrotransposon in the genome. The Ac element was present in McClintock’s strains. (b) Elements that move by retrotransposition (via an RNA intermediate) Transposase Catalyzes the Excision and Insertion of Transposons FI GURE 10.12 Common organization of DNA sequences in Now that we have considered the typical organization of TEs, let’s transposable elements. Direct repeats (DRs) are identical sequences examine the steps of the transposition process. The enzyme trans- found on both sides of all TEs. Inverted repeats (IRs) are at the ends of posase catalyzes the removal of a transposon from its original site in some transposable elements. Long terminal repeats (LTRs) are regions containing a large number of tandem repeats. the chromosome and its subsequent insertion at another location. A general scheme for simple transposition is shown in Figure 10.13a. 1. Transposase monomers first bind to the inverted repeat se- quences at the ends of the TE. Simple Transposons By comparison, a simple transposon 2. The monomers then dimerize, which brings the inverted re- carries one or more genes that are not required for transposition to peats close together. occur. For example, the simple transposon shown in Figure 10.12a 3. The DNA is cleaved between the inverted and direct re- carries an antibiotic-resistance gene. peats, which excises the TE from its original site within the chromosome. Retrotransposons The organization of retrotransposons varies 4. Transposase carries the TE to a new site and cleaves the greatly. They are categorized based on their evolutionary relation- target DNA sequence at staggered recognition sites. The ship to retroviruses. As described in Chapter 18, retroviruses are TE is then inserted into the target DNA and ligated to it. RNA viruses that make a DNA copy that integrates into the host’s genome. As shown in Figure 10.13b, the ligation of the transposable ele- LTR retrotransposons are evolutionarily related to retrovi- ment into its new site initially leaves short gaps in the target DNA. ruses. These TEs have retained the ability to move around the ge- Notice that the DNA sequences in these gaps are complementary nome, though, in most cases, they do not produce mature viral to each other (in this case, ATGCT and TACGA). Therefore, when particles. LTR retrotransposons are so named because they con- they are filled in by DNA gap repair synthesis, the repair produces tain long terminal repeats (LTRs) at both ends (Figure 10.12b). direct repeats that flank both ends of the TE. These direct repeats The LTRs are typically a few hundred base pairs in length. Like are common features found adjacent to all TEs (see Figure 10.12). their viral counterparts, LTR retrotransposons may code virally Although the transposition process depicted in Fig- related proteins, such as reverse transcriptase and integrase, that ure 10.13 does not directly alter the number of TEs, simple transposi- are needed for the retrotransposition process. tion is known to increase their numbers in genomes, in some cases to bro50795_ch10_237-269.indd 248 17/06/23 11:02 AM  10.5 Transposition 249 Transposable element Transposase cleaves the target DNA at staggered sites. 5′ 3′ T Inverted A T G C Target repeat Transposase subunits A T A C G DNA bind to inverted repeats. Transposase 3′ 5′ subunit The transposable element is inserted into the target site. The dimerization of transposase 5′ 3′ subunits causes the TE to loop out. T A T G C A T A C G 3′ 5′ Transposable element DNA gap repair synthesis 5′ 3′ T T Transposase cleaves outside of the A T G C A T G C A A inverted repeats (see pink arrows), T A C G T A C G which excises the transposon from 3′ 5′ the chromosomal DNA. Transposable Excised TE element Direct repeats (b) The formation of direct repeats Excised TE is inserted into a F I G URE 10. 1 3 Simple transposition. (a) new chromosomal location. Transposase removes the TE from its original site and inserts it into a new site. (b) A closer look at how the insertion process creates direct repeats. (a) Movement of a transposon via transposase Transposition TE fairly high levels. How can this happen? The answer is that transposi- tion often occurs around the time of DNA replication (Figure 10.14). After a replication fork has passed a region containing a TE, two TEs will be found behind the fork—one in each of the replicated regions. DNA replication proceeds past the TE One of these TEs could then transpose from its original location into point where the TE has been a region ahead of the replication fork. After the replication fork has inserted. The top copy of the TE passed this second region and DNA replication is completed, two then transposes ahead of the fork, where it is copied again. TEs will be found in one of the chromosomes and one TE in the other chromosome. In this way, simple transposition can lead to an TE increase in TE number. We will discuss the biological significance of transposon proliferation later in this section. TE TE Retrotransposons Use Reverse Transcriptase for Retrotransposition The bottom copy of DNA has 2 TEs. Thus far, we have considered how transposons can move through- F I G URE 1 0. 1 4 Increase in the number of copies of a trans- out a genome. By comparison, retrotransposons use an RNA inter- posable element (TE) via simple transposition. In this example, a TE mediate in their transposition mechanism. Let’s begin with LTR that has already been replicated transposes to a new site that has not yet retrotransposons. As shown in Figure 10.15, the movement of replicated. Following the completion of DNA replication, the TE has in- LTR retrotransposons requires two key enzymes: reverse creased in number. bro50795_ch10_237-269.indd 249 17/06/23 11:02 AM  250 C H A P T E R 1 0 :: MOLECULAR STRUCTURE OF CHROMOSOMES AND TRANSPOSABLE ELEMENTS 6. The retrotransposon DNA is then integrated into the target site. RNA Reverse 7. The gaps in the DNA are filled in, perhaps by DNA gap re- transcriptase Integrase pair synthesis, described in Chapter 19 (see Section 19.6). DNA Transposable Elements May Have Important Transcription Influences on Mutation and Evolution Over the past few decades, researchers have found that TEs prob- ably occur in the genomes of all species. Table 10.1 describes a few TEs that have been studied in great detail. As discussed earlier Ty in this chapter, the genomes of eukaryotic species typically con- tain moderately and highly repetitive sequences. In some cases, these repetitive sequences are due to the proliferation of TEs. In the genomes of mammals, for example, LINEs are long inter- Ty Ty Ty spersed elements that are usually 1000 to 10,000 bp in length and occur in 20,000 to 1,000,000 copies per genome. In humans, a FIGURE 10.15 Retrotransposition of an LTR retrotransposon. particular family of related LINEs