Molecular Biology
(Biochemistry of the Gene)
The Structural Basis of Cellular Information:
DNA, Chromosomes, and the Nucleus
 Cells possess a set of “instructions” that specify their structure, dictate their
functions, and regulate their activities
 These instructions can be passed on faithfully to daughter cells
 Hereditary information is transmitted in the form of distinct units called
genes
Genes consist of DNA
 Genes consist of DNA that codes for functional products that are usually
protein chains
 The information in a cell’s DNA molecules undergoes replication to generat
e two copies, for distribution into each daughter cell
Figure 18-1A
Transcription and translation
 Instructions stored in DNA are transmitted in a two-stage process, called
transcription and translation
 Transcription: RNA is synthesized in an enzymatic reaction that copies
information from DNA
 Translation: the base sequence of RNA is used to direct the synthesis of a
polypeptide
Figure 18-1B
Chemical Nature of the Genetic Material
 Walther Flemming first observed chromosomes under the microscope
 Just a few years before, Miescher reported the discovery of the substance
now known as DNA
Miescher’s Discovery of DNA Led to Conflicting Prop
osals Concerning the Chemical Nature of Genes
 Miescher extracted a material from white blood cells that he called “nuclei
n,” now called DNA
 He also extracted it from salmon sperm and believed it to be involved in
heredity
 However, because of inaccuracies in measurement of DNA in eggs and sperm,
he changed his mind
DNA and chromosomes
 In the early 1880s a botanist named Eduard Zacharias found that removing
DNA from cells abolished the staining of chromosomes
 He and others began to infer that DNA is the genetic material
 In the early 1900s, incorrectly interpreted staining experiments led to the
false conclusion that amounts of DNA in cells change dramatically
Genes and protein
 From 1910 to the 1940s most scientists believed that genes were made of
protein rather than DNA
 Proteins were thought to be more complex than DNA and thus more likely to
be the genetic material
 This idea prevailed until two important lines of evidence confirmed that DNA
is the genetic material
Avery Showed That DNA Is the Genetic Material
of Bacteria

F. Griffith, studying a pathogenic
bacterial strain that caused
pneumonia in animals, found two
forms of the bacterium

S-strain caused a fatal infection when
introduced into mice

R-strain was unable to do so
Figure 18-2A,B
Genetic transformation

When dead S-strain and living R-strain were mixed and used to infect
mice, the mice died

Griffith found many live S-strain bacteria in the dead mice

He concluded that the R-strain had been converted into S-strain, a
process called genetic transformation
Figure 18-2C, D, E
Avery and colleagues identified the
transforming substance

Oswald Avery and colleagues followed up the experiments of Griffiths by
trying to determine what the transforming substance was

They fractionated extracts of the S-strain bacteria and found that only the
nucleic acid fraction was able to transform the R-strain

Digesting the DNA from the extract prevented transformation
 Oswald Avery pursued the investigation of bacterial transformation by
asking which component of the heat-killed S bacteria was actually
responsible for the transforming activity
The fractionated cell-free fraction
DNA
Only the nucleic acid fraction
Causes transformation
Protein
We prove to be right !!!
HAHAHAHAHAHA
ㅎㅎㅎㅎㅎㅎ
ㅋㅋㅋㅋㅋㅋ
^^ ^^ ^^ ^^ ^^ ^^ ^^
It is lots of fun to blow
bubbles but it is wiser to
prick them yourself
before someone else
tries to !!!!
Life is not
fair !!!
Sometimes
luck is
more
important
than your
real ability
Hershey and Chase Showed That DNA Is the
Genetic Material of Viruses

Bacteriophages (or just phages)
are viruses that infect bacteria

Phage T2, which infects E. coli, is
one of the best studied

During infection the virus
attaches to the bacterial cell
surface and injects material into
the cell
Day Hershey and Martha Chase
Figure 18A-1
Figure 18A-2B
The genetic material of T2 phage
 Soon after infection, the bacterial cell begins to produce thousands of new
copies of the virus
 Hershey and Chase labeled phage proteins with radioactive sulfur, 35S, and
the DNA with radioactive phosphorus, 32P, and allowed the phages to infect
the bacteria
 In this way they could trace the fate of proteins and DNA during infection
The experiment
 After infection, once the material is injected into the bacteria, the empty
phage protein coats (ghosts) were removed by agitating cells in a blender
 Cells were recovered by centrifugation
 They then measured the radioactivity in the supernatant and the cells at the
bottom of the tube
Figure 18-3A
The results
 The results showed that most of the 32P remained with the bacterial cells,
but the majority of the 35S was found in the surrounding medium
 Hershey and Chase concluded that DNA and not protein had been injected
into the bacterial cells
 Therefore, DNA was the genetic material of the phage
Figure 18-3B
Chargaff’s Rules Reveal That A=T and
G=C
Chargaff’s Rules Reveal That A = T and G =
C
 Erwin Chargaff was interested in the base composition of DNA, and used
chromatographic methods to separate and quantify the relative amounts of
the four bases
 He showed that the DNA from different cells of a given species has the same
percentage of each of the four bases
 The base composition varies among species
Chargaff’s most striking observation
 Chargaff observed that for all DNA samples examined the number of A = the
number of T and the number of G = the number of C
 These are called Chargaff’s rules
 The significance was not understood until Watson and Crick proposed the
double-helix model for DNA structure
Table 18-1
My GC
content is
40 %
Mine
too
DNA Structure
 Once it was determined that DNA was the genetic material, a new set of
questions began to emerge
 One of the first was how cells are able to accurately replicate their DNA to be
passed on during cell division
 Answering this question required an understanding of the 3-D structure of
DNA
Watson and Crick Discovered That DNA Is
a Double Helix
 Watson and Crick built wire models to try to determine the structure of
DNA that agreed with everything known about DNA
 It was known that DNA had a sugar phosphate backbone with nitrogenous
bases attached to each sugar
 It was known that at physiological pH, the bases would be able to form
hydrogen bonds with each other
The double helix model
 The critical evidence came from X-ray diffraction data produced by Rosalind
Franklin
 It revealed that DNA was a long thin helical molecule
 Based on this information and other observations Watson and Crick
produced the double helix model
Watson and Crick Discovered that DNA
Is a Double Helix
After 40 Years
If it were not for my X-ray
diffraction photograph, James and
Francis would never discover the
structure of DNA.
I did not know that the other two
men were using my own
experimental findings. But I did
not complain
Rosalind Franklin
(1919 – 1956)
A victim of ovarian cancer
The double helix model (continued)
 In the double helix, the sugar-phosphate backbones are on the outside of
the helix with the bases on the inside, forming “steps” in a “spiral staircase”
 There are 10 nucleotide pairs per complete turn, and 0.34 nm per
nucleotide pair
 The 2-nm diameter of the helix is too small for purines and too large for
pyrimidines, but just right for one of each
The double helix model (continued)
 The purine-pyrimidine pairing is consistent with Chargaff’s rules
 The two strands are held together by hydrogen bonding between bases on
opposite strands
 The hydrogen bonds only fit within the helix when they form between
complementary bases: adenine with thymine and guanine with cytosine
Figure 18-4A
Figure 18-4B
Replication of genetic information
 The most important aspect of the double helix model was that it
suggested a mechanism for replication of DNA
 The two strands could separate so that each could act as a template to
dictate synthesis of a new complementary strand
Antiparallel strands
 The phosphodiester bonds that join the 5′ carbon of one nucleotide to the
3′ carbon of the next are oriented in opposite directions in the two DNA
strands
 This is called antiparallel orientation and has important implications for
both replication and transcription
Types of helix
 The right-handed helix is called B-DNA
 Naturally occurring B-DNA helices are flexible with variable shapes and
dimensions, depending on nucleotide sequence; it is the main form of DNA
 Z-DNA is a left-handed helix; its biological significance is not wellunderstood
Z-DNA is a lefthanded double
helix. Name Z
was derived
from the zig
zag pattern of
its sugarphosphate
backbone and
it’s longer and
thinner than BDNA
You can find me
where purines
are alternating
or pyrimidines
are alternating
or cytosine with
methyl groups
I’m right handed.
Shorter and thicker
than B DNA.
You can create me
artificially by
dehydrating B DNA
A-DNA (left), B-DNA (middle) and Z-DNA (right) -- 12 bp each
DNA Can Be Interconverted Between
Relaxed and Supercoiled Forms
 The DNA double helix can be twisted upon itself to form supercoiled DNA
 Positive supercoil: the DNA is twisted farther in the same direction as the
helix
 Negative supercoil: the DNA is twisted in the opposite direction as the helix
 Relaxed: no twisting of the DNA
Figure 18-6 (Top)
Figure 18-6 (Bottom)
Supercoiling and gene regulation
 Supercoiling affects both spatial organization and energy state of DNA, and
so affects the ability of the DNA to interact with other molecules
 Tighter winding of the double helix reduces the chances of interaction
 Negative supercoiling increases access to proteins involved in replication or
transcription
 Circular DNA molecules found in nature, including those of
bacteria, viruses and eukaryotic organelles are invariably
negatively supercoiled.
 Positive supercoiling  favors tighter winding of the double
helix
 Negative supercoling  tends to unwind the double helix
You can
enter
inside my
pants !!
Nothing
can
enter
inside
my
pants !!!
Positive Supercolied !!
Negative Supercolied !!
Interconversion between relaxed and
supercoiled DNA
 Topoisomerases can both induce and relax supercoils
 Type I topoisomerases: introduce transient single-strand breaks in DNA
 Type II topoisomerases: introduce double-stranded breaks; one example in
bacteria is DNA gyrase
Figure 18-7A
Figure 18-7B
The Two Strands of a DNA Double Helix Can Be
Separated Experimentally by Denaturation and
Rejoined by Renaturation
 DNA strands are bound together by relatively weak noncovalent bonds
 Strand separation (denaturation) can be induced experimentally by raising
temperature or pH
 The process can be monitored because single- and double-stranded DNA
differ in light absorption
Denaturation
 All DNA absorbs light with a maximum around
260 nm
 As the strands separate the absorbance increases rapidly
 The temperature at which half of the absorbance change is reached is
called the DNA melting temperature Tm; the Tm varies depending on A-T
content
Figure 18-8
The
temperature
at which
half of the
max
absorbance
has been
achieved
Melting points reflects how tightly the
DNA double helix is held together
 GC base pairs held by 3 hydrogen bonds (G≡C)
 AT base pairs held by 2 hydrogen bonds (A=T)
 So GC pairs are more resistant to separation than AT pairs
 The melting points therefore increase proportinal to the number of
GC pairs
Tm ∝ GC content
Figure 18-9
Renaturation
 Denatured DNA can be renatured by lowering the temperature to permit
hydrogen bonds to reform
 In nucleic acid hybridization, nucleic acids can be identified based on
sequence
 Denatured DNA is incubated with a purified single-stranded DNA (a probe)
with a sequence complementary to the sequence one is trying to detect
Figure 18-10
The Organization of DNA in Genomes
 The genome of an organism consists of the DNA that contains one
complete copy of all the genetic information of that organism
 A haploid set of chromosomes consists of one representative of each type
of chromosome whereas a diploid set contains two of each
Genome Size Generally Increases with an
Organism’s Complexity
 Genome size is usually expressed in base
pairs (bps)
 Kilobases (Kb, thousands), Megabases (Mb, millions), Gigabases (Gb;
billions) are used as abbreviations
 There is a wide range of genome sizes among organisms, which generally
increases with organism complexity
Figure 18-11
Restriction Endonucleases Cleave DNA
Molecules at Specific Sites
 Restriction endonucleases (restriction enzymes) cut DNA molecules at
specific internal sites
 The resulting DNA pieces are called restriction fragments
 The specific recognition sequence is called a restriction site
Figure 18B-1
Separation of Restriction Fragments by Gel
Electrophoresis
 A mixture of restriction fragments can be separated by gel electrophoresis
 DNA molecules are negatively charged and so will migrate toward the anode
 Small DNA molecules are separated in polyacrylamide gels; larger fragments
in
agarose gels
Figure 18-12
The submarine gel
Gel eletrophoresis. In a gel (either
agarose or polyacrylamide), the
negatively charged DNA fragments
move toward the positive electrode
at a rate inversely proportional to
their length. After the electric field
is applied for a certain period, DNA
fragments with different lengths will
be separated, which can be
visualized by autoradiography or by
treatment with a fluorescent dye (e.g.,
ethidium bromide). The relationship
between the size of a DNA fragment
and the distance it migrates in the
gel is logarithmic. Therefore, from
the band positions, the lengths of
DNA fragments can be determined.
You should stain me
before you see me !!!
Detection of DNA in gels
 DNA can be detected with ethidium bromide which binds DNA and
fluoresces orange under UV light
 If DNA fragments have been radioactively labeled, they can be detected by
autoradiography, using X-ray film to yield an autoradiogram
You
should
wear
gloves !!!
Restriction Mapping
 A researcher determines the order of restriction fragments of a DNA
molecule by treating the DNA with restriction enzymes followed by gel
electrophoresis
 DNA is cut with one restriction enzyme and with combinations of enzymes
to get the overall restriction map
Figure 18-13
Rapid Procedures Exist for DNA
Sequencing
 Two methods were devised for DNA sequencing, around the same time
restriction digesting was developed
 DNA sequencing: determining the linear order of bases in DNA
 The two methods were devised by Maxam and Gilbert, and Sanger and
colleagues
Sequencing methods
 The Maxam and Gilbert sequencing uses a chemical method, based on use
of nonprotein chemicals that cleave DNA preferentially at certain bases
 The Sanger procedure, or chain termination method, uses
dideoxynucleotides that when incorporated into a DNA chain, interfere with
further synthesis of DNA
Sanger sequencing has been adapted for use
in automated machines
 A single-stranded DNA template is used as a template to guide synthesis of
complementary DNA
 DNA synthesis is carried out in the presence of the deoxynucleotides, dATP,
dCTP, dGTP, dTTP
 These are the normal nucleotides incorporated into growing DNA chains
Sanger sequencing
 Small amounts of four dideoxynucleotides are added to the synthesis
reaction (ddATP, ddCTP, ddGTP, ddTTP) (1)
 Each of these is labeled with a different
fluorescent dye
 When a ddNTP is incorporated into the growing chain, synthesis ceases and
the DNA strand is labeled with the fluorescent tag of the ddNTP
 When a dideoxynucleotide is incorporated into a growing DNA chain in
place of the normal deoxynucleotide, DNA synthesis is prematurely
halted because the absence of 3’ hydroxyl group makes it impossible to
form a bond with the next nucleotide
Sanger sequencing—the result
 A mixture of DNA strands is produced by Sanger sequencing, each with a
fluorescent label attached that corresponds to the incorporated ddNTP (2)
 The sample is run on a polyacrylamide gel, which separates the DNA
strands based on size (3)
Figure 18-14
Sanger sequencing (continued)
 The fragments run through the gel with the smaller pieces running faster
 As they move through the gel, a special camera detects the color of each
fragment as it moves
past (4)
 In the automated machines this information is collected for hundreds of
bases in a row, and fed into a computer
The Genomes of Many Organisms Have
Been Sequenced
 DNA sequencing is now so commonplace and rapid that it is routinely
applied to entire genomes
 Computers are used to assemble sequences of short fragments into longer
stretches millions of bases in length
 Many organisms have had their genomes sequenced
The Genomes of Numerous Organisms
Have Been Sequenced
 Although DNA sequencing machines can only determine the
sequence of short pieces of DNA, usually 500 to 800 bases long,
one at a time, computer programs search for overlapping
sequences between such fragments and thereby allow data from
hundreds or thousands of DNA pieces to be assembled into
longer stretches that can reach millions of bases in length
Table 18-2
Completed in 2003, the Human Genome Project (HGP) was a 13-year project
coordinated by the U.S. Department of Energy and the National Institutes of
Health. During the early years of the HGP, the Wellcome Trust (U.K.) became a
major partner; additional contributions came from Japan, France, Germany,
China, and others.
Project goals were to:
identify all the approximately 20,000-25,000 genes in human DNA,
determine the sequences of the 3 billion chemical base pairs that make up human DNA,
store this information in databases,
improve tools for data analysis,
transfer related technologies to the private sector, and
address the ethical, legal, and social issues (ELSI) that may arise from the project.
The New Field of Bioinformatics Has Emerged to
Decipher Genomes and Proteomes
 Unraveling the sequence of bases was the ‘easy part’
Now comes the hard part
of figuring out the
meaning of this sequence
of 3 billion A’s, G’s, C’s
and T’s.
Which stretches of DNA
correspond to genes, when
and in what tissues are
these genes expressed,
what kind of proteins do
they code for ? How all
these proteins interact with
each other and
function ??????
The Field of Bioinformatics Has Emerged to
Decipher Genomes, Transcriptomes, and
Proteomes
 Unraveling the DNA sequence of a genome is easier than
determining
 What parts of the DNA correspond to genes
 What kinds of proteins the genes encode
 How the genes interact with each other
 How they function
 The field of bioinformatics merges computer science with
biology to address questions such as these
Humans may have twice number of
genes as do worms or flies..
+
=
Junk DNA ???
 Computer analyses also revealed
that only about 1 – 2 % of the
human genome actually
represents coding sequences.
 Although the remaining DNA
contains some important
regulatory elements, most of it
appears to consist of “junk” DNA
with no apparent function
 maybe not, see the mini RNA
that recently comes into focus
Transcriptomes
 The DNA sequence of an organism’s genome provides only partial
understanding of the functions of the genes
 A transcriptome is the entire set of RNA molecules produced by a genome
 DNA microarray technology facilitates the study of transcriptomes
Proteome
 The function of most genes is to
produce proteins, scientist are
beginning to look beyond the
genome to study the ‘proteome’the structure and properties of
every protein produced by a
genome
Mass spectometry
 Mass spectrometry is a high speed sensitive technique that separates
proteins or fragments based on differences in mass and charges
 Peptides can be produced by digesting proteins with specific proteases
 The peptides can be analyzed by mass spectrometry to identify them
Mass Spectrometry boosted proteome
research
Protein microarray
 It is possible to immobilize thousands of different proteins in tiny spots
on a small piece of glass
 The resulting protein microarrays can be used to study a variety of
protein properties, such as ability to bind other molecules
Analysis of large amount of data
 The huge amount of new data produced by these techniques must be
analyzed by computers
 One of the most widely used tools is BLAST (Basic Local Alignment Search
Tool),software that searches databases to locate DNA or protein sequences
that are similar to other known sequences
Tiny Differences in Genome Sequence
Distinguish People from One Another
 On average about 99.7% of the bases in one person’s genome will match the
published sequence of the human genome
 The remaining 0.3% of bases will vary from person to person, creating
features that make us unique individuals
 Single nucleotide polymorphisms (SNPs) are single-base differences
between individuals
Single nucleotide polymorphisms
 Most SNPs are not located in protein coding parts of genes
 It is not necessary to examine all 20 million SNPs individually because they
are not independent of one another
 SNPs close to each other on the same chromosome tend to be inherited
together in blocks called haplotypes
SNPs and the HapMap
 A database of haplotypes, the HapMap, provides a shortcut for scientists
trying to make a connection between a disease and certain genes.
 Once a trait has been linked to a haplotype, only the SNPs associated with it
are studied to determine which one is responsible
 DNA rearrangements also contribute to genome variability
Copy number variants
 There are DNA segments thousands of bases long present in variable numbers
of copies among individuals
 Each person’s genome is thought to contain hundreds of these copy number
variations (CNVs)
 These may involve millions of bases, overall
Repeated DNA Sequences Partially Explain the
Large Size of Eukaryotic Genomes
 In the 1960s Britten and Kohne discovered repeat DNA sequences
 They broke DNA into small fragments, denatured them by heating, and
allowed them to renature
 The rate of renaturation depends on the concentration of each kind of DNA
sequence—those found in high concentration reanneal more quickly
Bacterial vs. mammalian DNA
 When mammalian and bacterial DNA were tested, it was expected that
bacterial DNA, having fewer types of DNA sequences, should reanneal much
faster
 The results were not as expected; the calf DNA consisted of two classes of
sequences that renature at very different rates
 About 40% of the calf DNA renatures more rapidly than bacterial DNA
Repeated DNA sequences
 The more rapidly annealing sequences of the calf DNA contain repeated DNA
sequences that are present in multiple copies
 Eukaryotes have variable amounts of repeated DNA in their genomes; the
rest is nonrepeated DNA
 There are two categories of repeated DNA: tandemly repeated DNA and
interspersed
repeated DNA
Figure 18-15
Complexity of chromosomal DNA
DNA reassociation (renaturation)
Double-stranded DNA
Denatured,
single-stranded
DNA
k2
Slower, rate-limiting,
second-order process of
finding complementary
sequences to nucleate
base-pairing
Faster,
zippering
reaction to
form long
molecules
of doublestranded
DNA
Cot
The parameter controlling the reassociation reaction is:
DNA concentration (Co)
Time of Incubation (t)
Cot  represents Co x t
Cot1/2 the concentration and time required to proceed half association
The greater Cot1/2  slower reaction
Why does concentration matter ?
(short time)
(loooooooooo~~ong time)
Why does complexity matter ?
(short time)
(loooooooooo~~ong time)
DNA complexity ∝ Renaturation time
Renaturation time ∝(DNA concentration)-1
 DNA complexity is a function of DNA concentration (C0) and time (t)
DNA reassociation kinetics (for a single DNA species)
Cot1/2 = 1 / k2
k2 = second-order rate constant
Co = DNA concentration
t1/2 = time for half reaction
% DNA reassociated
log Cot
0
50
100
Cot1/2
Ideal second-order DNA reassociation curve (Cot curve)
Complexity expressed as base-pairs (bp)
100
1
101
102
103
2
104
105
106
3
107
4
108
109
1010
5
Cot1/2
10-6 10-5 10-4 10-3 10-2 10-1 100
Cot
1 = poly(dT)-poly(dA)
2 = purified human satellite DNA
3 = T4 bacteriophage DNA
4 = E. coli genomic DNA
5 = purified human single-copy DNA
101
102 103 104
There is a direct
relationship between
Cot1/2 and complexity
DNA reassociation kinetics for a mixture of DNA species
% DNA reassociated
Cot1/2 = 1 / k2
k2 = second-order rate constant
Co = DNA concentration
t1/2 = time for half reaction
0
50
100
fast (repeated)
intermediate
(repeated)
Cot1/2
Cot1/2
slow (single-copy)
Cot1/2
I
I
I
Kinetic fractions:
fast
intermediate
slow
I
I
I
log Cot
I
I
human genomic DNA
I
Tandemly Repeated DNA
• One major category of DNA repeats is called tandemly repeated DNA
• The multiple copies are arranged next to each other in a row
• It accounts for 10–15% of a typical mammalian genome; a repeat unit can
measure anywhere from 1 to 2000 bp, most of the time less than 10 bases
• Tandemly repeated DNA was originally called ‘satellite DNA’ (because its
distinctive base composition causes it to appear in a satellite band that
separates from the rest of the genomic DNA during centrifugation process)
Because AT base pair and GC base pair
differ slightly in their molecular weight
Simple-sequence repeats
 The tandem repeats that are less than 10 bases per repeat comprise a
subcategory called simple-sequence repeated DNA
 There can be as many as several hundred thousand copies at selected sites
in the genome
 It was originally called satellite DNA
Functions of simple-sequence repeats
 Such sequences are not usually transcribed, so they may be responsible for
imposing special physical properties on regions of the chromosome
 In eukaryotes, chromosomal regions called centromeres, with a role in
chromosome segregation, are rich in simple-sequence repeats
 Telomeres, at the ends of chromosomes, also contain simple-sequence
repeats
Figure 18-3, Part I
Minisatellites
 The amount of satellite DNA at any given site can vary enormously; typically
it ranges from 105 to 107 bp in overall length
 The term minisatellite is used to describe shorter regions, between 102 and
105 in length
 Microsatellites are even shorter, 10–100 bp in length, but with numerous
sites in the genome
DNA fingerprinting
 Microsatellite and minisatellite DNA is useful for DNA fingerprinting
 It uses gel electrophoresis to compare DNA fragments derived from different
genome regions
 It is a way to identify individuals
Figure 18C-1, Steps 1–3
Figure 18C-1, Steps 4, 5
Triplet repeat diseases
 Some diseases are traceable to excessive numbers of triplet repeats (triplet
repeat amplification), such as Huntington’s disease
 Other such diseases include fragile X syndrome, and myotonic dystrophy
Interspersed Repeated DNA
 Interspersed repeated DNAs are scattered around the genome
 Single repeats are hundreds or thousands of bases in length and the
dispersed copies, numbering in hundreds of thousands of copies, are similar
but not identical to one another
 They account for 25–50% of mammalian genomes
Types of interspersed repeated DNA
 Most interspersed repeated DNA consists of families of transposable
elements (transposons), which can move around the genome and leave
copies of themselves behind
 Roughly half of the human genome consists of these mobile elements
 The most abundant are called LINEs (Long interspersed nuclear elements)
LINEs and SINEs
 LINEs are 6000–8000 bp long and contain genes required for their own
mobilization
 SINEs are short interspersed nuclear elements and are less than 500 bp
 These rely on enzymes from other elements for their movement; the
most common SINEs in humans are Alu sequences, which account for 10%
of the human genome
Figure 18-16
DNA Packaging
 Very long molecules of DNA must be fit into the cell and in the case of
eukaryotes, into the nucleus
 DNA packaging is a challenge for all forms of life
Bacteria Package DNA in Bacterial
Chromosomes and Plasmids
 Bacterial chromosomes were once thought to be naked DNA
 However, it is now known that the DNA is packaged somewhat similarly to
the chromosomes of eukaryotes
 The main bacterial genome is called the bacterial chromosome
Bacterial Chromosomes
 Bacteria have single, multiple, linear or circular chromosomes depending on
the species but a single circular chromosome is most common
 The DNA molecule is bound to small amounts of protein and localized to a
region of the bacterial cell called the nucleoid
 The bacterial DNA is negatively supercoiled and folded into loops
Figure 18-17
Bacterial chromosomes
 The loops of bacterial DNA are held in place by RNA and basic protein
molecules
 Treatment with ribonuclease degrades RNA and releases some of the loops
 Nicking with a topoisomerase does not affect the loops but relaxes the
supercoils
Bacterial plasmids
 Besides the chromosome, bacteria may contain one or more plasmids, small
usually circular DNA molecules containing genes for their own replication
 They may also carry genes for cellular functions
 Most are supercoiled, and though they replicate autonomously, replication is
somewhat synchronous with the chromosome
Types of plasmids
 F (fertility) factors are involved in the process of conjugation
 R (resistance) factors carry genes that impart drug resistance to the
bacterium
 col (colinogenic) factors allow bacteria to secrete colicins, compounds that
kill nearby bacteria that lack the col factor
Types of plasmids (continued)
 Virulence factors enhance the ability to cause disease by producing toxic
proteins
 Metabolic plasmids produce enzymes required for certain metabolic
reactions
 Cryptic plasmids have no known function
Eukaryotes Package DNA in Chromatin
and Chromosomes
 In eukaryotes, more DNA is involved per cell and it interacts with more
proteins
 When bound to proteins, DNA is converted into chromatin
 At the time of division, the chromatin fibers condense into a more compact
structure, the chromosome
Histones
 Histones are a group of small basic proteins with high lysine and arginine
content
 The negatively charged DNA binds stably to the positively charged proteins
 The mass of histones in a chromosome is approximately equal to the mass of
the DNA
Types of histones
 There are five main types of histones, H1, H2A, H2B, H3, and H4
 Chromatin contains about equal numbers of all of these except H1, which is
present in about half the amount of the others
 Chromatin also contains a number of nonhistone proteins, which play a
variety of roles
Nucleosomes Are the Basic Unit of
Chromatin Structure
 In the 1960s X-ray diffraction studies revealed that chromatin has a repeating
structural subunit seen in neither DNA nor histones alone
 When isolated from cells, chromatin fibers appear as a series of tiny particles
attached by thin filaments (“beads-on-a-string”)
 The “beads” are called nucleosomes
Figure 18-18
Evidence for nucleosomes
 Chromatin can be exposed to a nuclease that cleaves DNA, and the partially
degraded DNA separated from proteins
 Electrophoresis shows a distinctive pattern of DNA bands in repeating 200
bp intervals
 This pattern is not generated when DNA alone is digested and it suggests
that nucleosomes occur at 200 bp intervals
Figure 18-19
Further evidence for nucleosomes
 Chromatin can be digested with micrococcal nuclease briefly
 The fragmented chromatin is then separated into fractions by centrifugation
 The smallest fraction contains a single spherical particle, the next fraction
contains two particles, and so on
 The particles are nucleosomes
Figure 18-20, Two upper panels
Figure 18-20, Two lower panels
A Histone Octamer Forms the
Nucleosome Core
 Kornberg and colleagues showed that nucleosomes can be assembled in
vitro only when the histones used were isolated gently
 Under these isolation procedures histone dimers H2A–H2B and H3–H4
remained intact
 They concluded that the H2A–H2B and H3–H4 complexes were an integral
part of the nucleosome
More investigation of the nucleosome
 Kornberg and colleagues used chemical crosslinking to show that
nucleosomes contain an octamer of eight histones
 Histone octamers contained two H2A–H2B dimers and two H3–H4 dimers,
with the DNA wrapped around the octamer
 The octomer with 146 bp of DNA is the core
particle; extra DNA from the original 200 bp is called linker DNA
More investigation of the nucleosome
 The amount of linker DNA varies among organisms, but the DNA
associated with the core particle always measures close to 146 bp
 This is enough DNA to wrap 1.7 times around the core particle
Figure 18-21
Histone H1
might be
associated
with the
linker region
Nucleosomes Are Packed Together
to Form Chromatin Fibers and Chromosomes
 Nucleosome formation is the first step in packaging of nuclear DNA
 Isolated chromatin (“beads on a string”) measures about 10 nm in diameter,
but chromatin of intact cells measures about 30 nm (the 30-nm chromatin
fiber)
 Histone H1 facilitates formation of the 30-nm fiber
Figure 18-22A, B
Further packing of chromatin
 The 30-nm fiber seems to be packed together in an irregular, threedimensional zigzag structure
 These fibers fold into looped domains 50,000–100,000 bp in length, attached
periodically to the chromosomal scaffold
 Chromatin so highly compacted that it shows up as dark spots on micrographs
is called heterochromatin; the more diffuse chromatin is called euchromatin
Figure 18-22B, C
Figure 18-23
Heterochromatin and euchromatin
 The tightly packed heterochromatin contains DNA that is transcriptionally
inactive
 The more diffuse, loosely packed euchromatin is associated with DNA that is
actively transcribed
 As a cell prepares to divide, all of its chromatin becomes highly compacted
 Because the chromosomal DNA has recently been duplicated, each
chromosome is composed of two chromatids
Figure 18-22D, E
Packing ratio
 The packing ratio is the extent to which a DNA molecule has
been folded (total length of DNA molecule/length of chromatin
fiber or chromosome)
 It is determined by determining the entire length of the DNA
molecule and dividing by the length of the fiber or
chromosome into which it has been packaged
 The packing ratio of DNA coiled around nucleosomes is around
7 and packing DNA into the 30-nm fiber results in a further
reduction, about six-fold
 Overall, the packing ratio of the 30-nm fiber is 42
DNA packaging ratio
= extended length of DNA molecule
/length of chromatin fiber
1
7
42
750
15,000 to 20,000
In chromosomes of
dividing cells
Eukaryotes Package Some of Their DNA in
Mitochondria and Chloroplasts
 Mitochondria and chloroplasts have their own chromosomes, that are
devoid of histones and are usually circular
 Though both organelles can encode some of their own polypeptides, they
are dependent on the nuclear genome to encode most of them
Figure 18-24
The human mitochondrion
 The genome of the human mitochondrion has been sequenced
 It is 16,569 base pairs long, and encodes 37 genes, about 5% of all the RNAs
and proteins needed by the mitochondrion
Figure 18-25
Mitochondrial genomes
 The size of mitochondrial genomes varies considerably among organisms
 Yeasts have mitochondrial genomes about 5X larger than those of mammals,
but most of the extra DNA is noncoding
 A 648-nucleotide sequence sometimes called the DNA bar code can be used
to distinguish closely related species
Chloroplast genomes
 Chloroplasts usually possess circular DNA molecules of about 120,000 bp in
length, containing around 120 genes
 Subunits of some multimeric protein complexes are encoded by the nuclear
genome; this is true for both chloroplasts and mitochondria
The Nucleus
 The nucleus is the site within the eukaryotic cell where the chromosomes
are localized and replicated and the DNA they contain is transcribed
 It is one of the most prominent and distinguishing features of eukaryotic
cells
Figure 18-26
Figure 18-27
A Double-Membrane Nuclear Envelope
Surrounds the Nucleus
 The nucleus is bounded by a nuclear envelope with an inner and an outer
membrane separated by a perinuclear space
 The outer membrane is continuous with the ER and contains proteins that
bind actin and IFs of the cytoskeleton
 Tubular invaginations of the envelope, the nucleoplasmic reticulum, project
into the nucleus
Nuclear pores
 Nuclear pores are specialized channels in the nuclear envelope, where inner
and outer membranes are fused
 They provide direct contact between the cytosol and the nucleoplasm
(interior nuclear space)
 They are lined with a protein structure called the nuclear pore complex (NPC)
Figure 18-28
Nuclear pore complex
 The NPC is built from about 30 different proteins called nucleoporins
 The complex has a striking symmetry
 The “central granule” is called the transporter, and is likely involved in
moving molecules across the nuclear envelope
Figure 18-29A
Figure 18-29B
Molecules Enter and Exit the Nucleus Through
Nuclear Pores
 Enzymes and proteins needed in the nucleus must be imported from the
cytoplasm
 RNAs that need to be translated and components of ribosomes must be
exported from the nucleus
 In addition to all this traffic through the pores, they also mediate passage of
small particles, molecules, and ions
Figure 18-30
Simple Diffusion of Small Molecules Through
Nuclear Pores
 Small particles, less than 10 nm in diameter, pass through pores at a rate
proportional to the size of the particle
 The NPC contains tiny aqueous diffusion channels through which small
particles freely move
Active Transport of Large Proteins and
RNA Through Nuclear Pores
 Some proteins needed in the nucleus are too large to easily diffuse through
the nuclear pores
 These large particles are actively transported across the membrane
 Nuclear localization signals (NLS) enable the protein to be recognized and
transported by the nuclear pore complex
The Import Process
 A cytoplasmic protein with
an NLS is recognized by a
receptor protein called an
importin, which binds the
NLS and mediates
movement of the protein to
a nuclear pore (1)
 The importin-protein
complex is transported into
the nucleus by the
transporter at the center of
the NPC (2)
 Inside the nucleus the importin
associates with a GTP-binding
protein called Ran, causing
importin to release the NLScontaining protein (3)
 The Ran-GTP importin complex is
transported back to the cytoplasm
through the NPC (4)
 In the cytoplasm the importin is
released as GTP is hydrolyzed (5)
The export process
 Export occurs by a comparable
process; RNA export is mediated
by adaptor proteins that
bind RNA
 The adaptor proteins bind to
nuclear export signals (NES);
NES sequences are recognized by
exportins, which mediate
transport of the complexes out
of the nucleus
Maintaining a Ran-GTP gradient across
the membrane
 Ran-GTP is maintained at high levels inside the nucleus by a guaninenucleotide exchange factor (GEF) that promotes Ran to bind GTP
 The cytosol contains a GTPase activating protein (GAP) that promotes
hydrolysis of GTP by Ran
Function of the Ran-GTP gradient across
the membrane
 The high nuclear Ran-GTP promotes the release of NLS-containing cargo from
importin
 It also promotes the binding of NES-containing cargo to exportin
 Nuclear transport factor 2 (NTF2) shuttles Ran-GDP back into the nucleus
The wife needs to come back home again
(to pay the taxi fare, you have money only at home)
The Nuclear Matrix and Nuclear Lamina
Are Supporting Structures of the Nucleus
 The nuclear matrix (nucleoskeleton) is an insoluble fibrous network that
helps maintain the shape of the nucleus
 The nuclear lamina is a thin dense meshwork of fibers lining the inner
surface of the inner nuclear membrane
 It is made of intermediate filaments made from lamins
Figure 18-32A
Figure 18-32B
Chromatin Fibers Are Dispersed Within the
Nucleus in a Nonrandom Fashion
 Most of the time a cell’s chromatin fibers are extended and dispersed
through the nucleus
 The chromatin of each chromosome has its own discrete location
(chromosome territory)
 In situ hybridization, using nucleic acid probes specific for sequences specific
to individual chromosomes, demonstrates this
Figure 18-33
Chromatin organization
 Parts of the chromatin are bound to the nuclear envelope near the nuclear
pores
 These regions are highly compacted, most of it constitutive
heterochromatin (highly condensed at all times; e.g., centromeres and
telomeres)
 Constitutive heterochromatin is composed of simple-sequence repeats
Facultative heterochromatin
 Facultative heterochromatin varies with the activities of the cell, and so can
differ from tissue to tissue
 It can even vary over time within one cell
The Nucleolus Is Involved in Ribosome
Formation
 The nucleolus is the place in the nucleus where ribosomal subunits are
assembled
 Fibrils in the nucleolus contain DNA that is being transcribed into ribosomal
RNA (rRNA)
 Granules in the nucleolus are rRNA molecules being packaged with
proteins
Figure 18-34
Figure 18-35
The NOR and nuclear bodies
 The nucleolus organizer region (NOR) is a stretch of DNA containing multiple
(hundreds to thousands) copies of rRNA genes
 Additional bodies in the nucleus play roles in processing and handling of RNA
molecules in the nucleus
 These are Cajal bodies, Gemini of Cajal bodies, speckles, and PML bodies