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Lecture Topic 13: DNA Organization in Eukaryotes

Welcome to Lecture Topic 13!

Please read and study the discussion below taken from Chapter 12 of our reference book. Also, watch the videos
which could supplement your understanding of the discussion.

Don't forget to answer the Quiz on Nov 2, 2023.

Introduction

We now turn our attention to the way DNA is organized in eukaryotic chromosomes, which are most clearly visible
as highly condensed structures during mitosis. However, after chromosome separation and cell division, cells enter
the interphase stage of the cell cycle, at which time the components of the chromosome uncoil and decondense into
a form referred to as chromatin. While in interphase, the chromatin is dispersed throughout the nucleus. As the cell
cycle progresses, cells may replicate their DNA and reenter mitosis, whereupon chromatin coils and condenses back
into visible chromosomes once again. This condensation represents a length contraction of some 10,000 times for
each chromatin fiber.

The organization of DNA during the transitions just described is much more intricate and complex than in viruses or
bacteria, which never exhibit a complex process similar to mitosis. This is due to the greater amount of DNA per
chromosome in eukaryotes, as well as the presence of a large number of proteins associated with eukaryotic DNA.
For example, while DNA in the E. coli chromosome is 1200 μm long, the DNA in each human chromosome
ranges from 19,000 to 73,000 μm in length. In a single human nucleus, all 46 chromosomes contain sufficient DNA
to extend almost 2 meters. This genetic material, along with its associated proteins, is contained within a nucleus
that usually measures about 5 to 10 μm in diameter.

Chromatin Structure and Nucleosomes

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The genetic material of viruses and bacteria consists of strands of DNA or RNA relatively devoid of proteins. In
contrast, eukaryotic chromatin has a substantial amount of protein associated with the chromosomal DNA in all
phases of the cell cycle. The associated proteins can be categorized as either positively charged histones or less
positively charged non-histone proteins. Of these two groups, the histones play the most essential structural
role. Histones contain large amounts of the positively charged amino acids lysine and arginine, making it possible
for them to bond electrostatically to the negatively charged phosphate groups of nucleotides. Recall that a similar
interaction has been proposed for several bacterial proteins. The five main types of histones are shown in Table 11.1.

Table 11.1 Categories and Properties of Histone Proteins

The general model for chromatin structure is based on the assumption that chromatin fibers, composed of DNA and
protein, undergo extensive coiling and folding as they are condensed within the cell nucleus. Moreover, X-ray
diffraction studies confirm that histones play an important role in chromatin structure. Chromatin produces
regularly spaced diffraction rings, suggesting that repeating structural units occur along the chromatin axis. If the
histone molecules are chemically removed from chromatin, the regularity of this diffraction pattern is disrupted.

A basic model for chromatin structure was worked out in the mid-1970s. The following observations were
particularly relevant to the development of this model:

1. Digestion of chromatin by certain endonucleases, such as micrococcal nuclease, yields DNA

fragments that are approximately 200 base pairs in length or multiples thereof. This enzymatic
digestion is not random, for if it were, we would expect a wide range of fragment sizes. Thus,
chromatin consists of some type of repeating unit, each of which protects the DNA from
enzymatic cleavage except where any two units are joined. It is the area between units that is
attacked and cleaved by the endonuclease.
2. Electron microscopic observations of chromatin have revealed that chromatin fibers are composed
of linear arrays of spherical particles (Figure 11.1). Discovered by Ada and Donald Olins, the particles
occur regularly along the axis of a chromatin strand and resemble beads on a string. These particles,
initially referred to as ν-bodies (ν is the Greek letter nu), are now called nucleosomes. These findings
conform to the above observation that suggests the existence of repeating units.
3. Studies of the chemical association between histone molecules and DNA in the nucleosomes of
chromatin show that histones H2A, H2B, H3, and H4 occur as two types of tetramers,
(H2A)2 · (H2B)2 and (H3)2 · (H4)2. Roger Kornberg predicted that each repeating nucleosome
unit consists of one of each tetramer (creating an octomer) in association with about 200 base pairs
of DNA. Such a structure is consistent with previous observations and provides the basis for a model
that explains the interaction of histones and DNA in chromatin.
4. When nuclease digestion time is extended, some of the 200 base pairs of DNA are removed from the
nucleosome, creating what is called a nucleosome core particle consisting of 147 base pairs. The
DNA lost in the prolonged digestion is responsible for linking nucleosomes together. This linker DNA
is associated with the fifth histone, H1.

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Figure 11.1 An electron micrograph revealing nucleosomes appearing as “beads on a string” along chromatin
strands derived from Drosophila melanogaster.

On the basis of this information, as well as on X-ray and neutron-scattering analyses of crystallized core particles
by John T. Finch, Aaron Klug, and others, a detailed model of the nucleosome was put forward in 1984, providing a
basis for predicting chromatin structure and its condensation into chromosomes. In this model, illustrated in Figure
11.2, a 147-bp length of the 2-nm-diameter DNA molecule coils around an octamer of histones in a left-handed
superhelix that completes about 1.7 turns per nucleosome. Each nucleosome, ellipsoidal in shape, measures about
11 nm at its longest point [Figure 11.2(a)]. Significantly, the formation of the nucleosome represents the first level of
packing, whereby the DNA helix is reduced to about one-third of its original length by winding around the histones.

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Figure 11.2 General model of the association of histones and DNA to form nucleosomes, illustrating the way in
which each thickness of fiber may be coiled into a more condensed structure, ultimately producing a metaphase
chromosome.

In the nucleus, the chromatin fiber seldom, if ever, exists in the extended form described in the previous
paragraph (that is, as an extended chain of nucleosomes). Instead, the 11-nm-diameter fiber is further packed into a
thicker structure, initially called a solenoid, but now referred to as a 30-nm fiber. [Figure 12.9(b)]. This thicker
structure, which is dependent on the presence of histone H1, consists of numerous nucleosomes coiled around and
stacked upon one another, creating a second level of packing. This provides a six-fold increase in compaction of the
DNA. It is this structure that is characteristic of an uncoiled chromatin fiber in interphase of the cell cycle. In the
transition to the mitotic chromosome, still further compaction must occur. The 30-nm structures are folded into a
series of looped domains, which further condense the chromatin fiber into a structure that is 300 nm in diameter
[Figure 12.9(c)]. These coiled chromatin fibers are then compacted into the chromosome arms that constitute a
chromatid, one of the longitudinal subunits of the metaphase chromosome [Figure 12.9(d)]. While Figure 12.9 shows
the chromatid arms to be 700 nm in diameter, this value undoubtedly varies among different organisms. At a value of
700 nm, a pair of sister chromatids comprising a chromosome measures about 1400 nm.

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The importance of the organization of DNA into chromatin and of chromatin into mitotic chromosomes can
be illustrated by considering that a human cell stores its genetic material in a nucleus about 5 to 10 mm in diameter.
The haploid genome contains more than 3 billion base pairs of DNA distributed among 23 chromosomes. The diploid
cell contains twice that amount. At 0.34 nm per base pair, this amounts to an enormous length of DNA (as stated
earlier, almost 2 meters)! One estimate is that the DNA inside a typical human nucleus is complexed with roughly 25
× 106 nucleosomes.

In the overall transition from a fully extended DNA helix to the extremely condensed status of the
mitotic chromosome, a packing ratio (the ratio of DNA length to the length of the structure containing it) of about
500 to 1 must be achieved. In fact, our model accounts for a ratio of only about 50 to 1. Obviously, the larger fiber can
be further bent, coiled, and packed to achieve even greater condensation during the formation of a mitotic
chromosome.

Chromatin Remodeling

As with many significant endeavors in genetics, the study of nucleosomes has answered some important
questions but at the same time has opened up new ones. For example, in the preceding discussion, we established
that histone proteins play an important structural role in packaging DNA into the nucleosomes that make up
chromatin. While this discovery helped solve the structural problem of how the huge amount of DNA is organized
within the eukaryotic nucleus, it brought another problem to the fore: When present in several levels of compaction
within the chromatin fiber, DNA is inaccessible to interaction with other important DNA-binding proteins. For
example, the various proteins that function in enzymatic and regulatory roles during the processes of replication and
transcription must interact directly with DNA. To accommodate these protein–DNA interactions, chromatin must be
induced to change its structure, a process now referred to as chromatin remodeling. To allow replication and gene
expression, chromatin must relax its compact structure and expose regions of DNA to these proteins, and there must
also be a mechanism for reversing the process during periods of inactivity.

Insights into how different states of chromatin structure might be achieved began to emerge in 1997, when Timothy
Richmond and members of his research team were able to significantly improve the level of resolution in X-
ray diffraction studies of nucleosome crystals, from 7 Å in the 1984 studies to 2.8 Å in the 1997 studies. One model
based on their work is shown in Figure 11.3. At this resolution, most atoms are visible, thus revealing the subtle
twists and turns of the superhelix of DNA encircling the histones. Recall that the double-helical ribbon in the figure
represents 147 bp of DNA surrounding four pairs of histone proteins. This configuration is essentially repeated over
and over in the chromatin fiber and is the principal packaging unit of DNA in the eukaryotic nucleus.

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Figure 11.3 The nucleosome core particle derived from X-ray crystal analysis at 2.8 Å resolution. The double-
helical DNA surrounds four pairs of histones.

By 2003, Richmond and colleagues achieved a resolution of 1.9 Å that revealed the details of the location of each
histone entity within the nucleosome. Of particular relevance to the discussion of chromatin remodeling is
the observation that there are unstructured histone tails that are not packed into the folded histone domains within
the core of the nucleosomes but instead protrude from it. For example, tails devoid of any secondary structure
extending from histones H3 and H2B protrude through the minor groove channels of the DNA helix. You should look
carefully at Figure 11.3 and locate examples of such tails. Other tails of histone H4 appear to make a connection with
adjacent nucleosomes. The significance of histone tails is that they provide potential targets along the chromatin
fiber for a variety of chemical modifications that may be linked to genetic functions, including chromatin remodeling
and the possible regulation of gene expression.

Several of these potential chemical modifications are now recognized as important to genetic function. One of the
most well-studied histone modifications involves acetylation by the action of the enzyme histone acetyltransferase
(HAT). The addition of an acetyl group to the positively charged amino group present on the side chain of the amino
acid lysine effectively changes the net charge of the protein by neutralizing the positive charge. Lysine is in
abundance in histones, and geneticists have known for some time that acetylation is linked to gene activation. It
appears that high levels of acetylation open up, or remodel, the chromatin fiber, an effect that increases in regions of
active genes and decreases in inactive regions. In the well-studied example of the inactivation of the X chromosome
in mammals, forming a Barr body, histone H4 is known to be greatly underacetylated.

Two other important chemical modifications are the methylation and phosphorylation of amino acids that are part of
histones. These chemical processes result from the action of enzymes called methyltransferases and kinases,
respectively. Methyl groups can be added to both arginine and lysine residues in histones, and this change has been
correlated with gene activity. Phosphate groups can be added to the hydroxyl groups of the amino acids serine and
histidine, introducing a negative charge on the protein. During the cell cycle, increased phosphorylation, particularly
of histone H3, is known to occur at characteristic times. Such chemical modification is believed to be related to the
cycle of chromatin unfolding and condensation that occurs during and after DNA replication. It is important to note
that the above chemical modifications (acetylation, methylation, and phosphorylation) are all reversible, under the
direction of specific enzymes.

Interestingly, while methylation of histones within nucleosomes is often positively correlated with gene activity in
eukaryotes, methylation of the nitrogenous base cytosine within polynucleotide chains of DNA, forming 5-
methyl cytosine, is usually negatively correlated with gene activity. Methylation of cytosine occurs most often when

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the nucleotide cytidylic acid is next to the nucleotide guanylic acid, forming what is called a CpG island. We must
conclude, then, that methylation can have a positive or a negative impact on gene activity.

The research described above has extended our knowledge of nucleosomes and chromatin organization and serves
here as a general introduction to the concept of chromatin remodeling. A great deal more work must be done,
however, to elucidate the specific involvement of chromatin remodeling during genetic processes. In particular, the
way in which the modifications are influenced by regulatory molecules within cells will provide important insights into
the mechanisms of gene expression. What is clear is that the dynamic forms in which chromatin exists are vitally
important to the way that all genetic processes directly involving DNA are executed. In addition, chromatin
remodeling is an important topic in the discussion of epigenetics, the study of modifications of an organism’s
genetic and phenotypic expression that are not attributable to alteration of the DNA sequence making up a gene.

Heterochromatin

Although we know that the DNA of the eukaryotic chromosome consists of one continuous double-helical fiber along
its entire length, we also know that the whole chromosome is not structurally uniform from end to end. In the early
part of the twentieth century, it was observed that some parts of the chromosome remain condensed and
stain deeply during interphase, while most parts are partially uncoiled and do not stain. In 1928, the terms
euchromatin and heterochromatin were coined to describe the parts of chromosomes that are uncoiled and those
that remain condensed, respectively.

Subsequent investigation revealed a number of characteristics that distinguish heterochromatin from

euchromatin. Heterochromatic areas are genetically inactive because they either lack genes or contain genes that
are repressed. Also, heterochromatin replicates later during the S phase of the cell cycle than does euchromatin. The
discovery of heterochromatin provided the first clues that parts of eukaryotic chromosomes do not always encode
proteins. For example, one particular heterochromatic region of the chromosome, the telomere, is thought to be
involved in maintenance of the chromosome’s structural integrity, and another region, the centromere, is involved in
chromosome movement during cell division.

The presence of heterochromatin is unique to and characteristic of the genetic material of eukaryotes. In some
cases, whole chromosomes are heterochromatic. A case in point is the mammalian Y chromosome, much of which
is genetically inert. The inactivated X chromosome in mammalian females is condensed into an inert
heterochromatic Barr body. In some species, such as mealy bugs, the chromosomes of one entire haploid set are
heterochromatic.

When certain heterochromatic areas from one chromosome are translocated to a new site on the same or another
nonhomologous chromosome, genetically active areas sometimes become genetically inert if they now lie adjacent
to the translocated heterochromatin. This influence on existing euchromatin is one example of what is
more generally referred to as a position effect. That is, the position of a gene or group of genes relative to all other
genetic material may affect their expression.

Chromosome Banding Differentiates Regions along the Mitotic Chromosome

Until about 1970, mitotic chromosomes viewed under the light microscope could be distinguished only by their
relative sizes and the positions of their centromeres. Unfortunately, even in organisms with a low haploid
number, two or more chromosomes are often visually indistinguishable from one another. Since that time,
however, cytological procedures were developed that made possible differential staining along the longitudinal axis
of mitotic chromosomes. Such methods are referred to as chromosome-banding techniques, because the
staining patterns resemble the bands of polytene chromosomes.

One of the first chromosome-banding techniques was devised by Mary-Lou Pardue and Joe Gall. They found that if
chromosome preparations from mice were heat denatured and then treated with Giemsa stain, a unique staining
pattern emerged: Only the centromeric regions of mitotic chromosomes took up the stain! The staining pattern was
thus referred to as C-banding. Relevant to our immediate discussion, this cytological technique identifies a specific
area of the chromosome composed of heterochromatin. A micrograph of the human karyotype treated in this way is
shown in Figure 11.4. Mouse chromosomes are all telocentric, thus localizing the stain at the end of each
chromosome.

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Figure 11.4 A human mitotic chromosome preparation processed to demonstrate C-banding. Only the centromeres
stain (as small dark circles).

Other chromosome-banding techniques were developed at about the same time. The most useful of these
techniques produces a staining pattern differentially along the length of each chromosome. This method, producing
G-bands (Figure 11.5), involves the digestion of the mitotic chromosomes with the proteolytic enzyme trypsin,
followed by Giemsa staining. The differential staining reactions reflect the heterogeneity and complexity of the
chromosome along its length.

Figure 11.5 G-banded karyotype of a normal human male. Chromosomes were derived from cells in metaphase.

In 1971 a uniform nomenclature for human chromosome-banding patterns was established based on G-
banding. Figure 11.6 illustrates the application of this nomenclature to the X chromosome. On the left of the
chromosome are the various organizational levels of banding of the p and q arms that can be identified; the resulting
designation for each of the specific regions is shown on the right side.

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Figure 11.6 The regions of the human X chromosome distinguished by its banding pattern. The designations on
the right identify specific bands.

Although the molecular mechanisms involved in producing the various banding patterns are not well understood, the
bands have played an important role in cytogenetic analysis, particularly in humans. The pattern of banding on each
chromosome is unique, allowing a distinction to be made even between those chromosomes that are identical in size
and centromere placement (e.g., human chromosomes 4 and 5 and 21 and 22). So precise is the banding pattern of
each chromosome that homologs can be distinguished from one another, and when a segment of one chromosome
has been translocated to another chromosome, its origin can be determined with great precision.

Eukaryotic Genomes Demonstrate Complex Sequence Organization Characterized by Repetitive DNA

Thus far, we have examined the general structure of chromosomes in eukaryotes. We now begin an examination of
what we know about the organization of DNA sequences within the chromosomes making up an organism’s genome.

In addition to single copies of unique DNA sequences that make up genes, many DNA sequences within
eukaryotic chromosomes are repetitive in nature. Various levels of repetition occur within the genomes of
organisms. Many studies have now provided insights into repetitive DNA, demonstrating various classes of
sequences and organization. Figure 11.7 schematizes these categories. Some functional genes are present in more
than one copy (they are referred to as multiple-copy genes) and so are repetitive in nature. However, the majority of
repetitive sequences do not encode proteins. Nevertheless, many are transcribed, and the resultant RNAs play
multiple roles in eukaryotes, including chromatin remodeling. We will explore three main categories of repetitive
sequences: (1) heterochromatin found to be associated with centromeres and making up telomeres, (2) tandem
repeats of both short and long DNA sequences, and (3) transposable sequences that are interspersed throughout the
genome of eukaryotes.

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Figure 11.7 An overview of the various categories of repetitive DNA.

Satellite DNA

The nucleotide composition (e.g., the percentage of G-C versus A-T pairs) of the DNA of a particular species
is reflected in the DNA’s density, which can be measured with a technique called sedimentation equilibrium
centrifugation, which in essence determines the molecule’s density. When eukaryotic DNA is analyzed in this way, the
majority is present and represented as a single main band, of fairly uniform density. However, one or more additional
peaks indicate the presence of DNA that differs slightly in density. This component, called satellite DNA, makes up a
variable proportion of the total DNA, depending on the species. For example, a profile of main-band and satellite DNA
from the mouse is shown in Figure 11.8. By contrast, bacteria do not contain satellite DNA.

Figure 11.8 Separation of main-band (MB) and satellite (S) DNA from the mouse by using ultracentrifugation in
a CsCl gradient.

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The significance of satellite DNA remained an enigma until the mid-1960s, when Roy Britten and David
Kohne developed a technique for measuring the re-association kinetics of DNA that had previously been dissociated
into single strands. The researchers demonstrated that certain portions of DNA reannealed more rapidly than others,
and concluded that rapid reannealing was characteristic of multiple DNA fragments composed of identical or
nearly identical nucleotide sequences—the basis for the descriptive term repetitive DNA. Recall that, in contrast,
bacterial DNA is nearly devoid of anything other than unique, single-copy sequences.

When satellite DNA is subjected to analysis by re-association kinetics, it falls into the category of highly repetitive
DNA, consisting of short sequences repeated a large number of times. Further evidence suggested that these
sequences are present as tandem (meaning adjacent) repeats clustered in very specific chromosomal areas
known to be heterochromatic—the regions flanking centromeres. This was discovered in 1969 when several
researchers, including Mary-Lou Pardue and Joe Gall, applied the technique of in situ hybridization to the study of
satellite DNA. This technique involves molecular hybridization between an isolated fraction of labeled DNA or RNA
probes and the DNA contained in the chromosomes of a cytological preparation. While fluorescent probes are
standard today, radioactive probes were used by Pardue and Gall. Following the hybridization procedure,
autoradiography was performed to locate the chromosome areas complementary to the fraction of DNA or RNA.

Pardue and Gall demonstrated that radioactive molecular probes made from mouse satellite DNA hybridize with DNA
of centromeric regions of mouse mitotic chromosomes (Figure 11.9). Several conclusions were drawn: Satellite DNA
differs from main-band DNA in its molecular composition, as established by buoyant density studies. It is
composed of short repetitive sequences. Finally, satellite DNA is found in the heterochromatic centromeric regions
of chromosomes.

Figure 11.9 In situ hybridization between a radioactive probe representing mouse satellite DNA and mouse mitotic
chromosomes. The grains in the autoradiograph are concentrated in chromosome regions referred to as
the centromeres, revealing them to consist of and represent the location of satellite DNA sequences.

Centromeric DNA Sequences

The separation of homologs during mitosis and meiosis depends on centromeres, described cytologically in the late
nineteenth century as the primary constrictions along eukaryotic chromosomes. In this role, it is believed that
the repetitive DNA sequences contained within the centromere are critical to this role. Careful analysis has confirmed
this belief. The minimal region of the centromere that supports the function of chromosomal segregation is
designated the CEN region. Within this heterochromatic region of the chromosome, the DNA binds a platform of
proteins, which in multicellular organisms includes the kinetochore that binds to the microtubules making up the
spindle fiber during division.

The CEN regions of the yeast Saccharomyces cerevisiae were the first to be studied. Each centromere serves an
identical function, so it is not surprising that CENs from different chromosomes were found to be remarkably similar
in their DNA sequence, displaying only minor differences between chromosomes. The CEN region of

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yeast chromosomes consists of about 120 bp. Mutational analysis suggests that portions near the 3' end of this
DNA region are most critical to centromere function since mutations in them, but not those nearer the 5' end, disrupt
centromere function. Thus, the DNA of this region appears to be essential to the eventual binding to the spindle fiber.

Centromere sequences of multicellular eukaryotes are much more extensive than in yeast and vary considerably in
size. For example, in Drosophila the CEN region is found embedded within some 200–600 kb of DNA, much of which
is highly repetitive (recall from our prior discussion that highly repetitive satellite DNA is localized in the centromere
regions of mice). In humans, one of the most recognized satellite DNA sequences is the alphoid family, found
mainly in the centromere regions. Alphoid sequences, each about 170 bp in length, are present in tandem arrays of
up to 1 million base pairs. It is now believed that such repetitive DNA in eukaryotes is transcribed and that the RNA
that is produced is ultimately involved in kinetochore function.

One final observation of interest is that the H3 histone, a normal part of most all eukaryotic nucleosomes, is
substituted by a variant histone designated CENP-A in centromeric heterochromatin. It is believed that the unique N-
terminal protein tails that make CENP-A unique are involved in the binding of kinetechore proteins that are essential
to the microtubules of spindle fibers. This finding supports the supposition that the DNA sequence found only in
centromeres is related to the function of this unique chromosomal structure.

Middle Repetitive Sequences: VNTRs and STRs

A brief look at still another prominent category of repetitive DNA sheds additional light on the organization of
the eukaryotic genome. In addition to highly repetitive DNA, which constitutes about 5 percent of the human
genome (and 10 percent of the mouse genome), a second category, middle (or moderately) repetitive DNA,
recognized by re-association kinetic studies, is fairly well characterized. Because we now know a great deal about
the human genome, we will use our own species to illustrate this category of DNA in genome organization.

Although middle repetitive DNA does include some duplicated genes (such as those encoding ribosomal RNA), most
prominent in this category are either noncoding tandemly repeated sequences or noncoding interspersed sequences.
No function has been ascribed to these components of the genome. An example is DNA described as variable
number tandem repeats (VNTRs). These repeating DNA sequences may be 15–100 bp long and are found within and
between genes. Many such clusters are dispersed throughout the genome, and they are often referred to as
minisatellites.

The number of tandem copies of each specific sequence at each location varies from one individual to the
next, creating localized regions of 1000–20,000 bp (1–20 kb) in length. The variation in size (length) of these regions
between individual humans was originally the basis for the forensic technique referred to as DNA fingerprinting.

Another group of tandemly repeated sequences consists of di-, tri-, tetra-, and pentanucleotides, also referred to as
microsatellites or short tandem repeats (STRs). Like VNTRs, they are dispersed throughout the genome and
vary among individuals in the number of repeats present at any site. For example, in humans, the most common
microsatellite is the dinucleotide (CA)n, where n equals the number of repeats. Most commonly, n is between 5 and
50. These clusters have served as useful molecular markers for genome analysis.

Repetitive Transposed Sequences: SINEs and LINEs

Still another category of repetitive DNA consists of sequences that are interspersed individually throughout the
genome, rather than being tandemly repeated. They can be either short or long, and many have the added
distinction of being transposable sequences, which are mobile and can potentially move to different locations within
the genome. A large portion of the human genome is composed of such sequences.

For example, short interspersed elements, called SINEs, are less than 500 base pairs long and may be
present 1,500,000 times or more in the human genome. The best characterized human SINE is a set of closely
related sequences called the Alu family (the name is based on the presence of DNA sequences recognized by the
restriction endonuclease Alu I). Members of this DNA family, also found in other mammals, are 200–300 base pairs
long and are dispersed rather uniformly throughout the genome, both between and within genes. In humans, the Alu
family encompasses more than 5 percent of the entire genome.

Alu sequences are of particular interest because some members of the Alu family are transcribed into RNA, although
the specific role of this RNA is not certain. Even so, the consequence of Alu sequences is their potential
for transposition within the genome, which is related to chromosome rearrangements during evolution. Alu

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sequences are thought to have arisen from an RNA element whose DNA complement was dispersed throughout the
genome as a result of the activity of reverse transcriptase (an enzyme that synthesizes DNA on an RNA template).

The group of long interspersed elements (LINEs) represents yet another category of repetitive transposable DNA
sequences. LINEs are usually about 6 kb in length and in the human genome are present approximately
850,000 times. The most prominent example in humans is the L1 family. Members of this sequence family are
about 6400 base pairs long and are present more than 500,000 times. Their 5' end is highly variable, and their role
within the genome has yet to be defined.

The general mechanism for transposition of L1 elements is now clear. The L1 DNA sequence is first transcribed into
an RNA molecule. The RNA then serves as the template for synthesis of the DNA complement using the
enzyme reverse transcriptase. This enzyme is encoded by a portion of the L1 sequence. The new L1 copy then
integrates into the DNA of the chromosome at a new site. Because of the similarity of this transposition mechanism
to that used by retroviruses, LINEs are referred to as retrotransposons.

SINEs and LINEs represent a significant portion of human DNA. SINEs constitute about 13 percent of the human
genome, whereas LINEs constitute up to 21 percent. Within both types of elements, repeating sequences of DNA are
present in combination with unique sequences.

Middle Repetitive Multiple-Copy Genes

In some cases, middle repetitive DNA includes functional genes present tandemly in multiple copies. For
example, many copies exist of the genes encoding ribosomal RNA. Drosophila has 120 copies per haploid genome.
Single genetic units encode a large precursor molecule that is processed into the 5.8S, 18S, and 28S rRNA
components. In humans, multiple copies of this gene are clustered on the p arm of the acrocentric chromosomes 13,
14, 15, 21, and 22. Multiple copies of the genes encoding 5S rRNA are transcribed separately from multiple clusters
found together on the terminal portion of the p arm of chromosome 1.

The Vast Majority of a Eukaryotic Genome Does Not Encode Functional Genes

Given the preceding information concerning various forms of repetitive DNA in eukaryotes, it is of interest to pose an
important question: What proportion of the eukaryotic genome actually encodes functional genes?

We have seen that, taken together, the various forms of highly repetitive and moderately repetitive DNA comprise
a substantial portion of the human genome—approximately 50 percent of all DNA sequences by most estimates. In
addition to repetitive DNA, a large amount of the DNA consists of single- copy sequences that appear to be
noncoding. Included are many instances of what we call pseudogenes. These are DNA sequences representing
evolutionary vestiges of duplicated copies of genes that have undergone significant mutational alteration. As a
result, although they show some homology to their parent gene, they are usually not transcribed because
of insertions and deletions throughout their structure.

While the proportion of the genome consisting of repetitive DNA varies among eukaryotic organisms, one
feature seems to be shared: Only a very small part of the genome actually codes for proteins. For example, the
20,000–30,000 genes encoding proteins in sea urchin occupy less than 10 percent of the genome. In Drosophila, only
5 to 10 percent of the genome is occupied by genes coding for proteins. In humans, it appears that the coding
regions of the estimated 20,000 functional genes occupy only about 2 percent of the total DNA sequence making up
the genome.

Study of the various forms of repetitive DNA has significantly enhanced our understanding of genome organization.

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QUIZ

Quiz for Lecture Topic 13

This is a short quiz to test if you have read and studied the discussions on this topic. You only have
2 minutes to submit your answers, otherwise they will not be recorded. Good luck!

Not attempted Due 2 November 2023

Chromosomes and DNA Packaging

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Histone Post Translational Modifications

Preparing Karyotypes, Staining, and Chromosomal Banding

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Human Genome - Repetitive DNA

An overview of the unexpressed regions of the human genome.

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