Genome Organization (PDF)

10.3 Organization of Functional Sites Along Eukaryotic Chromosomes 243 ∙∙ Origins of replication are chromosomal sites that are neces- 10.2 COMPREHENSION QUE...

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 called LINE-1, or L1, is found Ty is a retrotransposon found in yeast. in about 500,000 copies and represents about 17% of the total hu- CONCEPT CHECK: What is the function of reverse transcriptase? man DNA! By comparison, SINEs are short interspersed elements that are less than 500 bp in length. A specific example of a SINE is the Alu sequence, present in about 1 million copies in the human genome. About 10% of the human genome is composed of this transcriptase and integrase. In this example, the cell already con- particular TE. tains a retrotransposon known as Ty within its genome. LINEs and SINEs continue to proliferate in the human ge- 1. This retrotransposon is transcribed into RNA. nome, but at a fairly low rate. In about 1 live birth in 100, an Alu 2. In a series of steps, reverse transcriptase uses this RNA or an L1 (or both) sequence has been inserted into a new site in the as a template to synthesize a double-stranded DNA human genome. On rare occasions, a new insertion can disrupt a molecule. gene and cause phenotypic abnormalities. For example, new inser- 3. The LTRs at the ends of the double-stranded DNA are then tions of L1 or Alu sequences into particular genes have been recognized by integrase, which makes staggered cuts at a shown, on occasion, to be associated with diseases such as hemo- target site in the host chromosome and catalyzes the inser- philia, muscular dystrophy, and breast and colon cancer. tion of the TE into the site. The relative abundance of TEs varies widely among differ- ent species. As shown in Table 10.2, TEs can be quite prevalent The integration of a retrotransposon can occur at many locations in amphibians, mammals, and flowering plants, but tend to be within the genome. Figure 10.15 shows the integration occurring less abundant in simpler organisms such as bacteria and yeast. at two locations. Furthermore, because a single retrotransposon The biological significance of TEs in the evolution of prokaryotic can be copied into many RNA transcripts, retrotransposons may and eukaryotic species remains a matter of debate. According to accumulate rapidly within a genome. the selfish DNA hypothesis, TEs exist because they have charac- Though the mechanism of non-LTR retrotransposition is not teristics that allow them to multiply within the chromosomal entirely understood, one popular model for the replication and in- DNA of living cells. In other words, they resemble parasites in tegration of non-LTR retrotransposons is called target-site the sense that they inhabit a cell without offering any selective primed reverse transcription (TPRT). A simplified version of advantage to the organism. They can proliferate as long as they do this model is shown in Figure 10.16. not harm the organism to the extent that they significantly disrupt 1. The retrotransposon is first transcribed into RNA with a survival. polyA tail at the 3′ end. Alternatively, other geneticists have argued that transposi- 2. The target DNA site is recognized by an endonuclease, tional events are often deleterious. Therefore, TEs would be elimi- which may be coded by the retrotransposon. This endonu- nated from the genome by natural selection if they did not also clease recognizes a consensus sequence of 5′-TTTTA-3′, offer a compensating advantage. Several potential advantages and initially cuts just one of the DNA strands. have been suggested. For example, TEs may cause greater genetic 3. The polyA tail of the retrotransposon RNA binds to this variation by promoting recombination. In addition, bacterial TEs nicked site due to A-T base pairing. often carry an antibiotic-resistance gene that provides the organ- 4. Reverse transcriptase then uses the target DNA as a primer ism with a survival advantage. Researchers have also suggested and makes a DNA copy of the RNA, which is why the pro- that transposition may cause the insertion of exons from one gene cess is named target-site primed reverse transcription. into another gene, thereby producing a new gene with novel 5. An endonuclease makes a second cut in the other DNA function(s). This phenomenon, called exon shuffling, is described strand usually about 7–20 nucleotides away from the first cut. in Chapter 27. bro50795_ch10_237-269.indd 250 17/06/23 11:02 AM  Non-LTR retrotransposon Target DNA 5′ 3′ 5′ 3′ AAAA TAAAA TTTT ATTT T 3′ 5′ 3′ 5′ Target site is cut in one strand Transcription of retrotransposon by an endonuclease. Retrotransposon RNA with polyA tail 5′ 3′ TAAAA 5′ 3′ ATTT T AAAA 3′ 5′ First cut Retrotransposon RNA binds to the site due to A-T base pairing. 5′ 3′ TAAAA A 3′ 5′ T T TA A T AA 3′ 5′ Reverse transcriptase copies the RNA into DNA. 5′ 3′ TAAAA A 3′ 5′ T T TA A T AA Target-site primed reverse transcription 5′ 3′ The endonuclease cuts the other DNA strand. Second cut 5′ 3′ TAAAA A 3′ 5′ T T TA A T AA 5′ 3′ The RNA is degraded and the retrotransposon is integrated into the target site. 5′ 3′ TAAAA AAAA ATTT T TTTT 3′ 5′ Non-LTR retrotransposon at a new site This leaves gaps in the DNA that are filled in by DNA polymerase and ligase. 5′ 3′ TAAAA AAAA ATTT T TTTT 3′ 5′ Non-LTR retrotransposon at a new site FI GURE 10.16 A simplified model for retrotransposition of a non-LTR retrotransposon. 251 bro50795_ch10_237-269.indd 251 17/06/23 11:02 AM  252 C H A P T E R 1 0 :: MOLECULAR STRUCTURE OF CHROMOSOMES AND TRANSPOSABLE ELEMENTS TAB L E 10.1 Examples of Transposable Elements Element Type Approximate Length (bp) Description Bacterial IS1 Transposon 768 An insertion element that is commonly found in five to eight copies in E. coli. Tn10 Transposon 9300 One of many different bacterial transposons that carry an antibiotic-resistance gene. Tn951 Transposon 16,600 A transposon that provides bacteria with genes that allow them to metabolize lactose. Yeast Ty element Retrotransposon 6300 Found in S. cerevisiae in about 35 copies per genome. Fruit Fly P element Transposon 500–3000 A transposon that may be found in 30–50 copies in P strains of Drosophila. It is absent from M strains. Copia-like element Retrotransposon 5000–8000 One of a family of TEs found in Drosophila, which vary slightly in their lengths and sequences. Typically, each family member is found in about 5–100 copies per genome. Humans Alu sequence Retrotransposon 300 A SINE found in about 1,000,000 copies in the human genome. L1 Retrotransposon 6500 A LINE found in about 500,000 copies in the human genome. Plants Ac or Ds Transposon 4500 Ac is an autonomous transposon found in corn and other plant species. It carries a transposase gene. Ds is a nonautonomous version that lacks a functional transposase gene. Opie Retrotransposon 9000 A retrotransposon found in plants that is related to the copia-like elements found in animals. This controversy remains unresolved, but studies have shown are likely to be harmful. Usually, transposition is a relatively rare event that TEs can rapidly enter the genome of an organism and prolifer- that occurs only in a few individuals under certain conditions, such as ate quickly. In Drosophila melanogaster, for example, a TE known exposure to radiation. As described in Chapter 17, prokaryotes and eu- as a P element was probably introduced into this species in the karyotes have mechanisms that greatly decrease the movement of TEs. 1950s. Laboratory stocks of Drosophila collected prior to this time When transposition occurs at a high rate, it is likely to be do not contain P elements. Remarkably, in the last 60 years, the P detrimental. In Drosophila, M strain females lack P elements and element has expanded throughout Drosophila populations world- lack the ability to inhibit P element transposition. If these M strain wide. The only strains without the P element are laboratory strains females are crossed with males that contain numerous P elements collected prior to the 1950s. This observation underscores the sur- (P strain males), the egg cells allow the P elements to transpose at prising ability of TEs to infiltrate a population of organisms. a high rate. The resulting hybrid offspring exhibit a variety of ab- Transposable elements have a variety of effects on chromosome normalities, which include a high rate of sterility, mutation, and structure and gene expression (Table 10.3). Many of these outcomes TA B L E 10.3 TAB L E 10.2 Possible Consequences of Transposition Abundance of Transposable Elements in the Genomes of Selected Consequence Cause Species Chromosome Structure Percentage of the Total Species Genome Composed of TEs* Chromosome breakage Excision of a TE. Frog (Xenopus laevis) 77 Chromosomal rearrangements Homologous recombination between TEs located at different positions in the genome. Corn (Zea mays) 60 Gene Expression Human (Homo sapiens) 45 Mutation Incorrect excision of TEs. Mouse (Mus musculus) 40 Gene inactivation Insertion of a TE into a gene. Fruit fly (Drosophila melanogaster) 20 Alteration in gene regulation Transposition of a gene next to a regulatory Nematode (Caenorhabditis elegans) 12 sequence or the transposition of a Yeast (Saccharomyces cerevisiae) 4 regulatory sequence next to a gene. Bacterium (Escherichia coli) 0.3 Alteration in the exon content Insertion of exons into the coding of a gene sequence of a gene via TEs. This *In some cases, the abundance of TEs may vary somewhat among different strains of the same species. The values reported here are typical values. phenomenon is called exon shuffling. bro50795_ch10_237-269.indd 252 17/06/23 11:02 AM  10.6 Structure of Eukaryotic Chromosomes in Nondividing Cells 253 chromosome breakage. This deleterious outcome, which is called diameter. Therefore, the DNA in a eukaryotic cell must be folded hybrid dysgenesis, occurs because the P elements were able to and compacted to a staggering extent to fit inside the nucleus. In insert into a variety of locations in the genome. eukaryotic chromosomes, as in prokaryotic chromosomes, this is accomplished by the binding of many different proteins to the DNA. The DNA-protein complex found within eukaryotic chro- 10.5 COMPREHENSION QUESTIONS mosomes is termed chromatin. In recent years, it has become 1. Which of the following types of transposable elements rely on increasingly clear that the proteins bound to chromosomal DNA an RNA intermediate for transposition? are subject to change over the life of the cell. These changes in a. Insertion elements protein composition, in turn, affect the degree of compaction of b. Simple transposons the chromatin. As discussed in Chapter 17, non-coding RNA molecules also play a role in chromatin structure. In this section, c. Retrotransposons we will consider the structures of chromosomes during inter- d. All of the above phase—the period of the cell cycle that includes the G1, S, and G2 2. The function of transposase is phases. In Section 10.7, we will examine the additional compac- a. to recognize inverted repeats. tion that is necessary to produce the highly condensed chromo- b. to remove a TE from its original site. somes present during M phase. c. to insert a TE into a new site. d. all of the above. Linear DNA Wraps Around Histone Proteins 3. According to the selfish DNA hypothesis, TEs exist because to Form Nucleosomes, the Repeating Structural a. they offer the host a selective advantage. Unit of Chromatin b. they have characteristics that allow them to multiply within As in some archaea, the repeating structural unit within eukaryotic the chromosomal DNA of living cells. chromatin is the nucleosome. In eukaryotes, a nucleosome is a c. they promote the expression of certain beneficial genes. double-stranded segment of DNA wrapped around an octamer of d. all of the above. histone proteins, or histones (Figure 10.17a). ∙∙ Each octamer contains two copies each of four different 10.6 STRUCTURE OF histone proteins: H2A, H2B, H3, and H4. These are called the core histones. EUKARYOTIC ∙∙ Each of the histone proteins consists of a globular domain CHROMOSOMES IN and a flexible, charged amino terminus called an amino- NONDIVIDING CELLS terminal tail. ∙∙ The DNA is negatively supercoiled over the surface of an Learning Outcomes: octamer; it makes 1.65 negative superhelical turns around the 1. Define chromatin. octamer. Positively charged amino acids lysine and arginine 2. Describe the structures of (1) nucleosomes, (2) nucleosome in- in the histone proteins play a major role in binding to the neg- teractions according to the zigzag model, and (3) loop domains. atively charged phosphate groups along the DNA backbone. 3. Analyze Noll’s results, and explain how they support the ∙∙ The amount of DNA required to wrap around the histone beads-on-a-string model. octamer is 146 or 147 bp. 4. Describe how loop domains are formed according to the ∙∙ At its widest point, a single nucleosome is about 11 nm in loop extrusion model. diameter. 5. Describe the key features of a topologically interacting domain. ∙∙ In 1997, Timothy Richmond and colleagues determined 6. Explain the general strategy of chromosome conformation the structure of a nucleosome by X-ray crystallography capture methods. (Figure 10.17b). 7. Compare and contrast euchromatin and heterochromatin, The chromatin of eukaryotic cells displays a repeating pattern in and distinguish between constitutive heterochromatin and which the nucleosomes are connected by linker regions of DNA facultative heterochromatin. that vary in length from 20 to 100 bp, depending on the species 8. Explain the meaning of chromosome territory. and cell type. It has been suggested that the overall structure of 9. List the four levels of chromosome structure in a nondividing cell. connected nucleosomes resembles beads on a string. This struc- ture shortens the length of the DNA molecule about sevenfold. A distinguishing feature of eukaryotic cells is that their chromo- In addition to the core histones, another histone protein, H1, somes are located within a cellular compartment known as the is found in most eukaryotic cells and is called the linker histone. It nucleus. The DNA within a typical eukaryotic chromosome is a binds to the DNA in the linker region between nucleosomes and single, linear, double-stranded molecule that may be hundreds of helps to organize adjacent nucleosomes (Figure 10.17c). The linker millions of base pairs in length. If the DNA from a single set of hu- histone is less tightly bound to the DNA than are the core histones. man chromosomes were stretched from end to end, the length would In addition, nonhistone proteins bound to the linker region play a be over 1 meter! By comparison, most eukaryotic cells are only role in the organization and compaction of chromosomes, and their 10–100 µm in diameter, and the cell nucleus is only about 2–4 µm in presence may affect the expression of nearby genes. bro50795_ch10_237-269.indd 253 17/06/23 11:02 AM